Lipid formulated compositions and methods for inhibiting expression of Eg5 and VEGF genes

ABSTRACT

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA) in a liquid particle formulation, methods of using the compositions to inhibit the expression of the Eg5/KSP and VEGF, and methods of using the compositions to treat pathological processes mediated by Eg5/KSP and VEGF expression, such as endometrial cancer.

This application is the U.S. national phase of International PatentApplication No. PCT/US2011/059937, filed Nov. 9, 2011, which claims thebenefit of U.S. Provisional Application Nos. 61/411,761, filed Nov. 9,2010, and 61/493,197, filed Jun. 3, 2011, each of which is incorporatedby reference in its entirety.

FIELD OF THE INVENTION

This invention relates to administration of lipid formulatedcompositions containing double-stranded ribonucleic acid (dsRNA) toinhibit the expression of the human kinesin family member 11 (Eg5/KSP)and vascular endothelial growth factor (VEGF) genes, and the use of thecompositions to treat pathological processes mediated by Eg5/KSP andVEGF expression, such as cancer, e.g., endometrial cancer.

BACKGROUND OF THE INVENTION

The maintenance of cell populations within an organism is governed bythe cellular processes of cell division and programmed cell death.Within normal cells, the cellular events associated with the initiationand completion of each process is highly regulated. In proliferativedisease such as cancer, one or both of these processes may be perturbed.For example, a cancer cell may have lost its regulation (checkpointcontrol) of the cell division cycle through either the overexpression ofa positive regulator or the loss of a negative regulator, perhaps bymutation. Alternatively, a cancer cell may have lost the ability toundergo programmed cell death through the overexpression of a negativeregulator. Hence, there is a need to develop new chemotherapeutic drugsthat will restore the processes of checkpoint control and programmedcell death to cancerous cells.

One approach to the treatment of human cancers is to target a proteinthat is essential for cell cycle progression. In order for the cellcycle to proceed from one phase to the next, certain prerequisite eventsmust be completed. There are checkpoints within the cell cycle thatenforce the proper order of events and phases. One such checkpoint isthe spindle checkpoint that occurs during the metaphase stage ofmitosis. Small molecules that target proteins with essential functionsin mitosis may initiate the spindle checkpoint to arrest cells inmitosis. Of the small molecules that arrest cells in mitosis, thosewhich display anti-tumor activity in the clinic also induce apoptosis,the morphological changes associated with programmed cell death. Aneffective chemotherapeutic for the treatment of cancer may thus be onewhich induces checkpoint control and programmed cell death.Unfortunately, there are few compounds available for controlling theseprocesses within the cell. Most compounds known to cause mitotic arrestand apoptosis act as tubulin binding agents. These compounds alter thedynamic instability of microtubules and indirectly alter thefunction/structure of the mitotic spindle thereby causing mitoticarrest. Because most of these compounds specifically target the tubulinprotein which is a component of all microtubules, they may also affectone or more of the numerous normal cellular processes in whichmicrotubules have a role. Hence, there is also a need for agents thatmore specifically target proteins associated with proliferating cells.

Human kinesin family member 11, e.g., Eg5 or KSP, is one of severalkinesin-like motor proteins that are localized to the mitotic spindleand known to be required for formation and/or function of the bipolarmitotic spindle. There is a report of a small molecule that disturbsbipolarity of the mitotic spindle (Mayer, T. U. et. al. 1999. Science286(5441) 971-4, herein incorporated by reference). More specifically,the small molecule induced the formation of an aberrant mitotic spindlewherein a monoastral array of microtubules emanated from a central pairof centrosomes, with chromosomes attached to the distal ends of themicrotubules. The small molecule was dubbed “monastrol” after themonoastral array. This monoastral array phenotype had been previouslyobserved in mitotic cells that were immunodepleted of the Eg5 motorprotein. This distinctive monoastral array phenotype facilitatedidentification of monastrol as a potential inhibitor of Eg5. Indeed,monastrol was further shown to inhibit the Eg5 motor-driven motility ofmicrotubules in an in vitro assay. The Eg5 inhibitor monastrol had noapparent effect upon the related kinesin motor or upon the motor(s)responsible for golgi apparatus movement within the cell. Cells thatdisplay the monoastral array phenotype either through immunodepletion ofEg5 or monastrol inhibition of Eg5 arrest in M-phase of the cell cycle.However, the mitotic arrest induced by either immunodepletion orinhibition of Eg5 is transient (Kapoor, T. M., 2000. J Cell Biol 150(5)975-80). Both the monoastral array phenotype and the cell cycle arrestin mitosis induced by monastrol are reversible. Cells recover to form anormal bipolar mitotic spindle, to complete mitosis and to proceedthrough the cell cycle and normal cell proliferation. These data suggestthat an inhibitor of Eg5 which induced a transient mitotic arrest maynot be effective for the treatment of cancer cell proliferation.

Vascular endothelial growth factor (VEGF), also known as vascularpermeability factor, VPF) is a multifunctional cytokine that stimulatesangiogenesis, epithelial cell proliferation, and endothelial cellsurvival. VEGF can be produced by a wide variety of tissues, and itsoverexpression or aberrant expression can result in a variety disorders,including cancers and retinal disorders such as age-related maculardegeneration and other angiogenic disorders.

Therefore, there is a need to explore the use of compounds that modulateboth Eg5/KSP and VEGF expression to treat human disorders, e.g., cancer.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

Sequences of siRNA targeting VEGF, including AD-3133, are described inUS Patent Publication Nos. 2006/0094032 and 2006/0223770.

Sequences of siRNA targeting Eg5/KSP including AD-12115 are described inU.S. Pat. No. 7,718,629 and US Patent Publication No. 2011/0015250.

Lipid formulations, including DLinDMA comprising formulations, of siRNAtargeting VEGF including AD-3133 and siRNA targeting Eg5/KSP includingAD-12115 are described in US Patent Publication No. 2010/0087508) andInternational Publication WO 2009/111658.

Lipid formulations of VEGF targeting siRNA including AD-3133 and Eg5/KSPtargeting siRNA including AD-12115 are also described in US PublicationNo. 2010/0267806) and International Publication Nos. WO 2010/105209 andWO 2011/034798.

The contents of these applications are incorporated by reference for allpurposes. In particular, the sequences of the siRNA disclosed in theseapplications, e.g., Tables 1 and 2, are incorporated by reference forall purposes.

SUMMARY OF THE INVENTION

The present inventors have found that cancers associated with liverinvolvement, such as endometrial cancer, may be effectively treated byadministering a lipid particle formulation that includes a cationiclipid (such as DLinDMA), a neutral lipid (such as DPPC), a PEGylatedlipid (such as PEG2000-C-DMA), cholesterol, and a mixture of nucleicacids (such as a combination of an siRNA that targets Eg5/KSP and ansiRNA that targets VEGF). For example, the lipid particle may beALN-VSP02.

Thus, in one aspect, the present invention relates to a method oftreating endometrial cancer in a subject in need thereof, comprisingadministering to the subject once ever 2 weeks at least about 0.1 mg/kg(preferably at least 0.7 mg/kg) of a composition comprising DLinDMA,DPPC, PEG2000-C-DMA, cholesterol, an siRNA targeting Eg5/KSP, and ansiRNA targeting VEGF. (The dosage amounts are based on the total amountof siRNA per kg of body weight). For instance, the dosage can beselected from about 0.1, 0.2, 0.3, 0.4, 0.7, 1.0, 1.25, 1.5, 1.7, 2.0,3.0, and 6.0 mg/kg. In one embodiment, the subject has previously failedto respond to at least one cancer treatment.

In additional embodiments, the siRNA targeting Eg5/KSP is AD12115 andthe siRNA targeting VEGF is AD3133 (e.g., in a 1:1 molar ratio).

In one embodiment, about 1.0 mg/kg of the composition is administeredevery two weeks. In one embodiment, about 1.25 mg/kg of the compositionis administered every two weeks. In another embodiment, about 0.7 mg/kgof the composition is administered every two weeks.

In one embodiment, the composition is administered by intravenous (IV)infusion (such as, for example, for about 15 minutes once every twoweeks). In one preferred embodiment, the intravenous infusion isperformed over 15 minutes to 1 hour (e.g., over 30, 45, or 60 minutes).

In another embodiment, the present invention relates to a method ofadministering an siRNA targeting Eg5/KSP and an siRNA targeting VEGF ina subject in need thereof, comprising administering to the subject atleast 0.1 mg/g of a composition comprising DLinDMA, DPPC, PEG2000-C-DMA,cholesterol, an siRNA targeting Eg5/KSP, and an siRNA targeting VEGF viaintravenous (IV) infusion once every 2 weeks, wherein the compositionhas a dose-proportional maximum concentration (C_(max)) and area undercurve (AUC) as measureable in the subject's plasma. In one embodiment,the subject is suffering form a disorder treatable with an siRNAtargeting Eg5/KSP and/or an siRNA targeting VEGF.

In one embodiment, the present invention relates to a method of treatingendometrial cancer in a subject in need thereof, comprisingadministering to the subject once ever 2 weeks a dosage of about 1.0mg/kg. In another embodiment, the present invention relates to a methodof treating endometrial cancer in a subject in need thereof, comprisingadministering to the subject once ever 2 weeks a dosage of about 1.25mg/kg. In one embodiment, the subject has previously failed to respondto at least one cancer treatment.

In one embodiment, the present invention relates to a method of treatingcancer (e.g, endometrial cancer) in a subject in need thereof,comprising administering to the subject a composition comprising ansiRNA targeting Eg5/KSP and an siRNA targeting VEGF (such as thosedescribed herein, including ALN-VSP02) that provides one or more of thepharmacokinetic parameters shown in Table 18 below for a 1.0 mg/kgdosage. In another embodiment, the present invention relates to a methodof treating cancer (e.g, endometrial cancer) in a subject in needthereof, comprising administering to the subject a dosage form thatprovides one or more of the pharmacokinetic parameters shown in Table 18for a 1.25 mg/kg dosage.

In one embodiment, the present invention relates to a method of treatingcancer (e.g, endometrial cancer) in a subject in need thereof,comprising administering to the subject a dosage form that provides oneor more of the pharmacokinetic parameters shown in Table 19 for a 1.0mg/kg dosage. In another embodiment, the present invention relates to amethod of treating cancer (e.g, endometrial cancer) in a subject in needthereof, comprising administering to the subject a dosage form thatprovides one or more of the pharmacokinetic parameters as shown in Table19 for a 1.25 mg/kg dosage.

In additional embodiments, the AUC_(0-last) of KSP siRNA following thefirst administration of 1 mg/kg of the composition to a subject iswithin about 80% to about 125% of 15393.10±6002.12 ng*hr/mL. Inadditional embodiments, the AUC_(0-last) of KSP siRNA following thesecond administration of 1 mg/kg of the composition to a subject iswithin about 80% to about 125% of 9984.02±2517.30 ng*hr/mL.

In additional embodiments, the C_(max) of KSP siRNA following the firstadministration of 1 mg/kg of the composition to a subject is withinabout 80% to about 125% of 15314.98±5347.86 ng/mL. In additionalembodiments, the C_(max) of KSP siRNA following the secondadministration of 1 mg/kg of the composition to a subject is withinabout 80% to about 125% of 12440.80±9615.15 ng/mL.

In additional embodiments, the AUC_(0-last) of VEGF siRNA following thefirst administration of 1 mg/kg of the composition to a subject iswithin about 80% to about 125% of 16404.33±7280.14 ng*hr/mL. Inadditional embodiments, the AUC_(0-last) of VEGF siRNA following thesecond administration of 1 mg/kg of the composition to a subject iswithin about 80% to about 125% of 9563.98±2173.22 ng*hr/mL.

In additional embodiments, the C_(max) of VEGF siRNA following the firstadministration of 1 mg/kg of the composition is within about 80% toabout 125% of 15843.73±7014.77 ng/mL. In additional embodiments, theC_(max) of VEGF siRNA following the second administration of 1 mg/kg ofthe composition is within about 80% to about 125% of 12681.94±8811.54ng/mL.

In additional embodiments, the AUC_(0-last) of KSP siRNA following thefirst administration of 1.25 mg/kg of the composition to a subject iswithin about 80% to about 125% of 33929.01±18334.75 ng*hr/mL. Inadditional embodiments, the AUC_(0-last) of KSP siRNA following thesecond administration of 1.25 mg/kg of the composition to a subject iswithin about 80% to about 125% of 28833.57 ng*hr/mL.

In additional embodiments, the C_(max) of KSP siRNA following the firstadministration of 1.25 mg/kg of the composition to a subject is withinabout 80% to about 125% of 21937.49±7642.00 ng/mL. In additionalembodiments, the C_(max) of KSP siRNA following the secondadministration of 1.25 mg/kg of the composition to a subject is withinabout 80% to about 125% of 17897.05 ng/mL.

In additional embodiments, the AUC_(0-last) of VEGF siRNA following thefirst administration of 1.25 mg/kg of the composition to a subject iswithin about 80% to about 125% of 33442.95±18558.98 ng*hr/mL. Inadditional embodiments, the AUC_(0-last) of VEGF siRNA following thesecond administration of 1.25 mg/kg of the composition to a subject iswithin about 80% to about 125% of 31072.98 ng*hr/mL.

In additional embodiments, the C_(max) of VEGF siRNA following the firstadministration of 1.25 mg/kg of the composition is within about 80% toabout 125% of 21578.94±6798.07 ng/mL. In additional embodiments, theC_(max) of VEGF siRNA following the second administration of 1.25 mg/kgof the composition is within about 80% to about 125% of 18230.74 ng/mL.

The present inventors have also found that co-administration of acorticosteroid and/or using a slower infusion rate during IVadministration diminishes the occurrence of adverse infusion reactionsfor the subject during administration of the compositions describedherein.

Thus, in a further embodiment, the methods described herein furthercomprise administering a corticosteroid to the subject prior to orconcurrently with the composition. In yet further embodiments, theduration of each IV infusion ranges from about 15 minutes to about 1hour or from about 30 minutes to about 1 hour, or is about 30 minutes,45 minutes, about 60 minutes.

Yet another embodiment is a method for treating a subject in needthereof comprising administering to the subject a dosage of acomposition comprising DLinDMA, DPPC, PEG2000-C-DMA, cholesterol, ansiRNA targeting Eg5/KSP, and an siRNA targeting VEGF (such as acomposition comprising ALN-VSP02) via intravenous (IV) infusion onceevery 2 weeks. Yet another embodiment is a method for treating a subjectin need thereof comprising administering to the subject a compositioncomprising DLinDMA, DPPC, PEG2000-C-DMA, cholesterol, an siRNA targetingEg5/KSP, and an siRNA targeting VEGF (such as a composition comprisingALN-VSP02), where the composition is administered via intravenousinfusion in a dosage selected from at least 0.1, 0.2, 0.3, 0.4, 0.7,1.0, 1.25, 1.5, 1.7, 2.0, 3.0, and 6.0 mg/kg. In one preferredembodiment, the dosage is about 1.0 mg/kg. In another embodiment, thedosage is about 1.25 mg/kg. In yet another embodiment, the dosage isabout 1.5 mg/kg. The subject may suffer from cancer, such as advancedcancer with liver involvement.

Another embodiment of the invention is a method for treating a subjectin need of treatment comprising administering to the subject a dosage ofa composition comprising ALN-VSP02 via intravenous (IV) infusion onceevery 2 weeks.

Yet another embodiment is a method for treating a subject in need oftreatment comprising administering via intravenous infusion ALN-VSP02 ina dosage selected from about 0.1, 0.2, 0.3, 0.4, 0.7, 1.0, 1.25, 1.5,1.7, 2.0, 3.0 and 6.0 mg/kg. In one preferred embodiment, the dosage isabout 1.0 mg/kg. In another embodiment, the dosage is about 1.25 mg/kg.In yet another embodiment, the dosage is about 1.5 mg/kg.

In one embodiment, the present invention relates to a method of treatingcancer in a subject in need thereof, comprising administering to thesubject once ever 2 weeks a dosage of about 1.0 mg/kg. In anotherembodiment, the present invention relates to a method of treating cancerin a subject in need thereof, comprising administering to the subjectonce ever 2 weeks a dosage of about 1.25 mg/kg.

The dosage forms administered can provide the C_(max) and AUC_(0-last)values for KSP and VEGF siRNA at the specified doses above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing liver weights as percentage of body weightfollowing administration of SNALP-siRNAs in a Hep3B mouse model.

FIGS. 2A-2D are graphs showing the effects of SNALP-siRNAs on bodyweight in a Hep3B mouse model.

FIG. 3 is a graph showing the effects of SNALP-siRNAs on body weight ina Hep3B mouse model.

FIG. 4 is a graph showing the body weight in untreated control animals.

FIG. 5 is a graph showing the effects of control luciferase-SNALP siRNAson body weight in a Hep3B mouse model.

FIG. 6 is a graph showing the effects of VSP-SNALP siRNAs on body weightin a Hep3B mouse model.

FIG. 7A is a graph showing the effects of SNALP-siRNAs on human GAPDHlevels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 7B is a graph showing the effects of SNALP-siRNAs on serum AFPlevels as measured by serum ELISA in a Hep3B mouse model.

FIG. 8 is a graph showing the effects of SNALP-siRNAs on human GAPDHlevels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 9 is a graph showing the effects of SNALP-siRNAs on human KSPlevels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 10 is a graph showing the effects of SNALP-siRNAs on human VEGFlevels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11A is a graph showing the effects of SNALP-siRNAs on mouse VEGFlevels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11B is a set of graphs showing the effects of SNALP-siRNAs on humanGAPDH levels and serum AFP levels in a Hep3B mouse model.

FIGS. 12A-12C are graphs showing the effects of SNALP-siRNAs on tumorKSP, VEGF and GAPDH levels in a Hep3B mouse model.

FIG. 13A and FIG. 13B are graphs showing the effects of SNALP-siRNAs onsurvival in mice with hepatic tumors. Treatment was started at 18 days(FIG. 13A) and 26 days (FIG. 13B) after tumor cell seeding.

FIG. 14 is a graph showing the effects of SNALP-siRNAs on serum alphafetoprotein (AFP) levels.

FIG. 15A and FIG. 15B are images of H&E stained sections in tumorbearing animals (three weeks after Hep3B cell implantation) wereadministered 2 mg/kg SNALP-VSP (A) or 2 mg/kg SNALP-Luc (B). Twenty fourhours later, tumor bearing liver lobes were processed for histologicalanalysis. Arrows indicate mono asters.

FIG. 16 is a flow diagram illustrating the manufacturing process ofALN-VSPDS01.

FIG. 17 is a cryo-transmission electron microscope (cryo-TEM) image ofALN-VSP02.

FIG. 18 is a flow diagram illustrating the manufacturing process ofALN-VSP02.

FIG. 19 is a graph illustrating the effects on survival ofadministration SNALP formulated siRNA and Sorafenib.

FIG. 20 is a graph illustrating the effect of ALN-VSP treatment of anintraperitoneal HCC mouse model on mitotic figure (monoaster) formationin tumors.

FIG. 21 is a graph illustrating the effect of ALN-VSP treatment of miceimplanted intrahepatically with HCT116 (colorectal carcinoma cells) onhKSP mRNA levels in tumors.

FIG. 22 are graphs illustrating the effect of ALN-VSP treatment of miceimplanted intrahepatically with HCT116 (colorectal carcinoma cells) onformation of mitotic figures (e.g., monasters) in liver (FIG. 22A) andlymph nodes (FIG. 22B).

FIG. 23 are graphs illustrating the effect of ALN-VSP treatment of micebearing orthotopic Hep3B tumors on tumor hemorrhage (FIG. 23A) and tumormicrovessel density (FIG. 23B).

FIG. 24 are graphs illustrating the effect of SNALP-VEGF only treatmentof mice bearing orthotopic Hep3B tumors on tumor hemorrhage (FIG. 24A)and tumor microvessel density (FIG. 24B).

FIG. 25 are graphs illustrating the effect of ALN-VSP treatmentmonoaster formation mouse models of intrahepatic colorectal carcinomatumors. Results are shown for liver (FIG. 25A), lung (FIG. 25B), lymphnodes and subcutaneous metastases (FIG. 25C).

DETAILED DESCRIPTION OF THE INVENTION

The invention provide methods of treatment, e.g., methods of treatmentof liver cancer, via IV infusion administration of a lipid formulatedcomposition having two siRNAs, one targeting the Eg5/KSP gene, and onetargeting the VEGF gene. In some embodiments the composition isALN-VSP02, as described herein. The composition can be administered onceevery two weeks, e.g., once every two weeks for at least 8 weeks. Thecomposition can be administered at a dosage of at least 0.4 mg/kg or atleast 0.7 mg/kg. The described method of treatment is well tolerated inpatients. Measurement of siRNA concentrations in plasma afteradministration of the composition shows dose proportional Cmax and AUCwith no evidence of drug accumulation.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. In another example, adenine and cytosine anywherein the oligonucleotide can be replaced with guanine and uracil,respectively to form G-U Wobble base pairing with the target mRNA.Sequences comprising such replacement moieties are embodiments of theinvention.

As used herein, “Eg5” refers to the human kinesin family member 11,which is also known as KIF11, Eg5, HKSP, KSP, KNSL1 or TRIP5. Eg5sequence can be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeqID number:NM_004523. The terms “Eg5” and “KSP” and “Eg5/KSP” are usedinterchangeably.

As used herein, vascular endothelial growth factor (VEGF), also known asvascular permeability factor, is an angiogenic growth factor. VEGF is ahomodimeric 45 kDa glycoprotein that exists in at least three differentisoforms. VEGF isoforms are expressed in endothelial cells. The VEGFgene contains 8 exons that express a 189-amino acid protein isoform. A165-amino acid isoform lacks the residues encoded by exon 6, whereas a121-amino acid isoform lacks the residues encoded by exons 6 and 7.VEGF145 is an isoform predicted to contain 145 amino acids and to lackexon 7. VEGF can act on endothelial cells by binding to an endothelialtyrosine kinase receptor, such as Flt-1 (VEGFR-1) or KDR/flk-1(VEGFR-2). VEGFR-2 is expressed in endothelial cells and is involved inendothelial cell differentiation and vasculogenesis. A third receptor,VEGFR-3, has been implicated in lymphogenesis.

The various isoforms have different biologic activities and clinicalimplications. For example, VEGF145 induces angiogenesis and like VEGF189(but unlike VEGF165) VEGF145 binds efficiently to the extracellularmatrix by a mechanism that is not dependent on extracellularmatrix-associated heparin sulfates. VEGF displays activity as anendothelial cell mitogen and chemoattractant in vitro and inducesvascular permeability and angiogenesis in vivo. VEGF is secreted by awide variety of cancer cell types and promotes the growth of tumors byinducing the development of tumor-associated vasculature Inhibition ofVEGF function has been shown to limit both the growth of primaryexperimental tumors as well as the incidence of metastases inimmunocompromised mice.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the Eg5/KSP and/or VEGF gene, including mRNA that is a product of RNAprocessing of a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding Eg5/KSP and/or VEGF) including a 5′ UTR, anopen reading frame (ORF), or a 3′ UTR. For example, a polynucleotide iscomplementary to at least a part of a Eg5 mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding Eg5.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aduplex structure comprising two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker”. The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA minus any overhangs that are present in the duplex. Inaddition to the duplex structure, a dsRNA may comprise one or morenucleotide overhangs. In general, the majority of nucleotides of eachstrand are ribonucleotides, but as described in detail herein, each orboth strands can also include at least one non-ribonucleotide, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, “dsRNA” may include chemical modifications toribonucleotides, including substantial modifications at multiplenucleotides and including all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an siRNA typemolecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule. In some embodiments the dsRNA can have anucleotide overhang at one end of the duplex and a blunt end at theother end.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches may be in the internal or terminal regions ofthe molecule. Generally the most tolerated mismatches are in theterminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA agent or aplasmid from which an iRNA agent is transcribed.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of” “down-regulate theexpression of,” “suppress the expression of” and the like in as far asthey refer to the Eg5 and/or VEGF gene, herein refer to the at leastpartial suppression of the expression of the Eg5 and/or VEGF gene, asmanifested by a reduction of the amount of Eg5 mRNA and/or VEGF mRNAwhich may be isolated from a first cell or group of cells in which theEg5 and/or VEGF gene is transcribed and which has or have been treatedsuch that the expression of the Eg5 and/or VEGF gene is inhibited, ascompared to a second cell or group of cells substantially identical tothe first cell or group of cells but which has or have not been sotreated (control cells). The degree of inhibition is usually expressedin terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Eg5 and/or VEGFgene expression, e.g. the amount of protein encoded by the Eg5 and/orVEGF gene which is produced by a cell, or the number of cells displayinga certain phenotype, e.g. apoptosis. In principle, target gene silencingcan be determined in any cell expressing the target, eitherconstitutively or by genomic engineering, and by any appropriate assay.However, when a reference is needed in order to determine whether agiven dsRNA inhibits the expression of the Eg5 and/or VEGF gene by acertain degree and therefore is encompassed by the instant invention,the assay provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of the Eg5 and/or VEGF issuppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% by administration of the double-stranded oligonucleotide of theinvention. In some embodiments, the Eg5 and/or VEGF gene is suppressedby at least about 60%, 70%, or 80% by administration of thedouble-stranded oligonucleotide of the invention. In other embodiments,the Eg5 and/or VEGF gene is suppressed by at least about 85%, 90%, or95% by administration of the double-stranded oligonucleotide of theinvention. The Example below provides values for inhibition ofexpression using various Eg5 and/or VEGF dsRNA molecules at variousconcentrations.

As used herein in the context of Eg5 and/or VEGF the terms “treat”,“treatment”, and the like, refer to relief from or alleviation ofpathological processes mediated by Eg5 and/or VEGF expression. In thecontext of the present invention insofar as it relates to any of theother conditions recited herein below (other than pathological processesmediated by Eg5 and/or VEGF expression), the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition, such as the slowing and progression of hepaticcarcinoma.

As used herein, the phrases “therapeutically effective amount” refers toan amount that provides a therapeutic benefit in the treatment ormanagement of pathological processes mediated by Eg5 and/or VEGFexpression or an overt symptom of pathological processes mediated by Eg5and/or VEGF expression. The specific amount that is therapeuticallyeffective can be readily determined by ordinary medical practitioner,and can vary depending on factors known in the art, such as, e.g. thetype of pathological processes mediated by Eg5 and/or VEGF expression,the patient's history and age, the stage of pathological processesmediated by Eg5 and/or VEGF expression, and the administration of otheranti-pathological processes mediated by Eg5 and/or VEGF expressionagents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological or therapeutic result. For example, if a given clinicaltreatment is considered effective when there is at least a 25% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 25%reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. As described in more detailbelow, such carriers include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.The term specifically excludes cell culture medium. For drugsadministered orally, pharmaceutically acceptable carriers include, butare not limited to pharmaceutically acceptable excipients such as inertdiluents, disintegrating agents, binding agents, lubricating agents,sweetening agents, flavoring agents, coloring agents and preservatives.Suitable inert diluents include sodium and calcium carbonate, sodium andcalcium phosphate, and lactose, while corn starch and alginic acid aresuitable disintegrating agents. Binding agents may include starch andgelatin, while the lubricating agent, if present, will generally bemagnesium stearate, stearic acid or talc. If desired, the tablets may becoated with a material such as glyceryl monostearate or glyceryldistearate, to delay absorption in the gastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

The term “ALN-VSP02” refers to a SNALP formulated composition comprisingtwo siRNAs: an siRNA targeting Eg5/KSP (AD12115), and an siRNA targetingVEGF (AD3133). A detailed description of ALN-VSP02 is described below inExample 9. The sequence of each ALN-VSP02 siRNA is as follows:

Duplex ID Target Sense (5′ to 3′) Antisense (5′ to 3′) AD12115 Eg5/KSPucGAGAAucuAAAcuAAcuTsT AGUuAGUUuAGAUUCUCGATsT (SEQ ID NO: 1)(SEQ ID NO: 2) AD3133 VEGF GcAcAuAGGAGAGAuGAGCUsUAAGCUcAUCUCUCCuAuGuGCusG (SEQ ID NO: 3) (SEQ ID NO: 4) Key: A, G, C,U-ribonucleotides; c, u-2′-O-Me ribonucleotides; s-phosphorothioate.

The SNALP formulation is as follows:

Proportion Component (mg/mL) AD12115 and AD3133  2.0* DLinDMA(1,2-Dilinoleyloxy-N,N-dimethyl-3- 7.3 aminopropane), DPPC(R-1,2-Dipalmitoyl-sn-glycero-3-phosphocholine) 1.1 Cholesterol,Synthetic 2.8 PEG2000-C-DMA (3-N-[(ω-Methoxy poly(ethylene glycol) 0.82000) carbamoyl]-1,2-dimyristyloxy-propylamine) Phosphate BufferedSaline (PBS) q.s.

The corresponding mol % for the proportions described in this table areas follows: 57.1/7.1/34.4/1.4 (DLinDMA/DPPC/Cholesterol/PEG2000-C-DMA).

As used herein, the term “intravenous (IV) infusion” refers to a methodof administration of a composition directly into the vein of a patient.IV infusion allows for direct administration of a composition to thebloodstream of a patient. This can be performed, for example, viasubcutaneous or intradermal infusion. IV infusion can be performed inmany ways, including through the use of an injection needle, or possiblywith an infusion pump. It can be provided as, for example, a continuousinfusion, an intermittent infusion, a patient-controlled infusion, or acircadian infusion.

As used herein, the term “Area Under the Curve” or “AUC” refers to theoverall amount of drug in the bloodstream after a dose. It is calculatedas the integral of the plasma drug concentration after the drug isadministered. AUC is obtained through collecting blood samples amultiple time points after administering a dose of a composition until atime where the amount of composition in the plasma is negligible.

The term “C_(max)” refers to the peak plasma concentration of acomposition after administration of the composition.

It is to be understood that the disclosure of any particularpharmacokinetic value (including C_(max) and AUC_(0-last)) includesdisclosure of a range of 80% to 125% of the value.

As used herein, the term “dose-proportional” describes a quantity whichhas a linear relationship with the amount of a composition administeredto a patient, i.e., the magnitude of increase or decrease of a quantitydependent on dosage is about the same as the dosage. For example, if adosage increase of about 2-fold correlates with an AUC increase of about2-fold, then the AUC is dose-proportional.

As used herein, the term “plasma” refers to the fluid portion of theblood in which particulate components, e.g. ALN-VSP02 composition, aresuspended. It can be obtained by sedimentation or centrifugation of theblood. Plasma represents approximately 50% of the total volume of bloodand contains glucose, proteins, amino acids, and other nutritivematerials; urea and other excretory products; and hormones, enzymes,vitamins, minerals, etc.

The term “blood plasma concentration” or “plasma concentration” refersto the concentration of a composition, such as ALN-VSP02 or either ofthe dsRNA of ALN-VSP02, in the plasma component of blood of a subject orpatient population. It is understood that the plasma concentration of acomposition, such as ALN-VSP02, may vary significantly between subjects,due to variability with respect to metabolism and/or possibleinteractions with other therapeutic agents.

Double-Stranded Ribonucleic Acid (dsRNA)

As described in more detail below, the invention providesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of the Eg5 and/or VEGF gene in a cell or mammal, and methodsof treatment using the dsRNA. The dsRNA comprises an antisense strandcomprising a region of complementarity which is complementary to atleast a part of an mRNA formed in the expression of the Eg5 and/or VEGFgene, and wherein the region of complementarity is less than 30nucleotides in length, generally 19-24 nucleotides in length, andwherein said dsRNA, upon contact with a cell expressing said Eg5 and/orVEGF gene, inhibits the expression of said Eg5 and/or VEGF gene.

ALN-VSP02 is a lipid formulated composition that includes two dsRNA, onetargeting Eg5/KSP and one targeting VEGF. The sequences of two dsRNAsare as follows:

Duplex ID Target Sense (5′ to 3′) Antisense (5′ to 3′) AD12115 Eg5/KSPucGAGAAucuAAAcuAAcuTsT AGUuAGUUuAGAUUCUCGATsT (SEQ ID NO: 1)(SEQ ID NO: 2) AD3133 VEGF GcAcAuAGGAGAGAuGAGCUsUAAGCUcAUCUCUCCuAuGuGCusG (SEQ ID NO: 3) (SEQ ID NO: 4) Key: A, G, C,U-ribonucleotides; c,u-2′-O-Me ribonucleotides; s-phosphorothioate.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. Additional synthesis methods are described below.

Additional siRNA, e.g., dsRNA targeting Eg5 and/or VEGF are alsocontemplated. Sequences of siRNA targeting VEGF, including AD-3133, aredescribed in U.S. patent application Ser. No. 11/078,073 filed Mar. 11,2005 (US Patent publication no 2006-0094032) and US continuation-in-partpatent application Ser. No. 12/754,110, filed Jan. 25, 2006 (US patentapplication publication no. 2006-0223770). Sequences of siRNA targetingEg5/KSP including AD-12115 are described in U.S. patent application Ser.No. 11/694,215 filed Mar. 30, 2007 (now U.S. Pat. No. 7,718,629). Thecontents of these applications are incorporated by reference for allpurposes. In particular, the sequences of the siRNA disclosed in theseapplications, e.g., Tables 1 and 2, are incorporated by reference forall purposes.

Additional dsRNA can be designed and described as follows.

The dsRNA comprises two strands that are sufficiently complementary tohybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) comprises a region of complementarity that issubstantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of the Eg5 and/or VEGF gene, the other strand (the sensestrand) comprises a region which is complementary to the antisensestrand, such that the two strands hybridize and form a duplex structurewhen combined under suitable conditions. Generally, the duplex structureis between 15 and 30, more generally between 18 and 25, yet moregenerally between 19 and 24, and most generally between 19 and 21 basepairs in length. In other embodiments the duplex structure is 25-30 basepairs in length.

In one embodiment the duplex is 19 base pairs in length. In anotherembodiment the duplex is 21 base pairs in length. When two differentsiRNAs are used in combination, the duplex lengths can be identical orcan differ. For example, a composition can include a first dsRNAtargeted to Eg5 with a duplex length of 19 base pairs and a second dsRNAtargeted to VEGF with a duplex length of 21 base pairs.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30, more generally between 18 and 25, yet more generallybetween 19 and 24, and most generally between 19 and 21 nucleotides inlength. In other embodiments the region of complementarity is 25-30nucleotides in length. In one embodiment, the region of complementarityis 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 24 nucleotides in length.

In one embodiment the region of complementarity is 19 nucleotides inlength. In another embodiment the region of complementarity is 21nucleotides in length. When two different siRNAs are used incombination, the region of complementarity can be identical or candiffer. For example, a composition can include a first dsRNA targeted toEg5 with a region of complementarity of 19 nucleotides and a seconddsRNA targeted to VEGF with a region of complementarity of 21nucleotides.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, or 24 nucleotides inlength. In other embodiments, each is strand is 25-30 base pairs inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ. Forexample, a composition can include a dsRNA targeted to Eg5 with a sensestrand of 21 nucleotides and an antisense strand of 21 nucleotides, anda second dsRNA targeted to VEGF with a sense strand of 21 nucleotidesand an antisense strand of 23 nucleotides.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4,generally 1 or 2 nucleotides. In another embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In further embodiments, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties than the blunt-ended counterpart. In someembodiments the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. A dsRNA having only one overhang has proven particularlystable and effective in vivo, as well as in a variety of cells, cellculture mediums, blood, and serum. Generally, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs can have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

As described in more detail herein, the composition of the inventionincludes a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF.The first and second dsRNA can have the same overhang architecture,e.g., number of nucleotide overhangs on each strand, or each dsRNA canhave a different architecture. In one embodiment, the first dsRNAtargeting Eg5 includes a 2 nucleotide overhang at the 3′ end of eachstrand and the second dsRNA targeting VEGF includes a 2 nucleotideoverhang on the 3′ end of the antisense strand and a blunt end at the 5′end of the antisense strand (e.g., the 3′ end of the sense strand).

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as wellIt can bereasonably expected that shorter dsRNAs comprising one of the sequencesof ALN-VSP02 minus only a few nucleotides on one or both ends may besimilarly effective as compared to the dsRNAs described above. Hence,dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20,or more contiguous nucleotides from one of the sequences of ALN-VSP02,and differing in their ability to inhibit the expression of the Eg5and/or VEGF gene in am assay as described herein by not more than 5, 10,15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence,are contemplated by the invention. Further dsRNAs that cleave within thetarget sequences of ALN-VSP02 can readily be made using the ALN-VSP02sequences and the target sequence provided.

In addition, ALN-VSP02 identifies a site in the Eg5 mRNA and a site inthe VEGF gene that is susceptible to RNAi based cleavage. As such thepresent invention further includes RNAi agents, e.g., dsRNA, that targetwithin the sequence targeted by ALN-VSP02. As used herein a second RNAiagent is said to target within the sequence of a first RNAi agent if thesecond RNAi agent cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first RNAi agent. Such asecond agent will generally consist of at least 15 contiguousnucleotides from one of the sequences of ALN-VSP02 to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in the Eg5/KSP and/or VEGF gene.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the Eg5 gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the Eg5/KSP and/or VEGF gene. Consideration of theefficacy of dsRNAs with mismatches in inhibiting expression of theEg5/KSP and/or VEGF gene is important, especially if the particularregion of complementarity in the Eg5/KSP and/or VEGF gene is known tohave polymorphic sequence variation within the population.

Modifications

The sequences of ALN-VSP02 include chemical modifications. In otherembodiments, the Eg5/KSP and/or VEGF targeting dsRNA used in the methodsinclude the same primary sequences as ALN-VSP02, but either nomodifications, a subset of the modifications of the sequences ofALN-VSP02, and/or additional modifications. Additional dsRNA targetingEg5/KSP and/or VEGF with sequences found in the patent applicationscited herein can be used in the described methods. These dsRNA can bechemically modified.

In some embodiments, the dsRNA is further chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Specific examples of preferred dsRNAcompounds useful in this invention include dsRNAs containing modifiedbackbones or no natural internucleoside linkages. As defined in thisspecification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Preferred modified dsRNA backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or ore or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an dsRNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta., 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

The dsRNAs may also include nucleobase (often referred to in the artsimply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosine's, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., DsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Conjugates

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199,86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994 4 1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan etal., Ann N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of an dsRNA compound. These dsRNAs typically contain atleast one region wherein the dsRNA is modified so as to confer upon thedsRNA increased resistance to nuclease degradation, increased cellularuptake, and/or increased binding affinity for the target nucleic acid.An additional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxy dsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim Biophys. Acta, 1995,1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterolmoiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative United States patents that teach the preparation of suchdsRNA conjugates have been listed above. Typical conjugation protocolsinvolve the synthesis of dsRNAs bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with thedsRNA still bound to the solid support or following cleavage of thedsRNA in solution phase. Purification of the dsRNA conjugate by HPLCtypically affords the pure conjugate.

In some cases, a ligand can be multifunctional and/or a dsRNA can beconjugated to more than one ligand. For example, the dsRNA can beconjugated to one ligand for improved uptake and to a second ligand forimproved release.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier and methods of administering the same. Thepharmaceutical composition containing the dsRNA is useful for treating adisease or disorder associated with the expression or activity of aEg5/KSP and/or VEGF gene, such as pathological processes mediated byEg5/KSP and/or VEGF expression, e.g., liver cancer. Such pharmaceuticalcompositions are formulated based on the mode of delivery.

Dosage

The composition, e.g., ALN-VSP02, is administered in a dosage sufficientto inhibit expression of Eg5/KSP and/or VEGF genes. Unless describedotherwise, dosage refers to the dose of total dsRNA. If more than onedsRNA is administered at the same time, dosage refers to the dosage ofboth dsRNA. For example, ALN-VSP02 includes two different dsRNA; adosage of ALN-VSP02 refers to the total dosage of both dsRNA.

In general, a suitable dose of dsRNA will be in the range of 0.01 to200.0 milligrams dsRNA per kilogram body weight of the recipient perday, generally in the range of 1 to 50 mg per kilogram body weight perday. In some embodiments a suitable dose of dsRNA is in the ranges of0.1 to 2.0 mg/kg.

For example, the dsRNA can be administered at a dosage of about 0.1,about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about0.8, about 0.9, about 1.0, about 1.05, about 1.1, about 1.15, about 1.2,about 1.25, about 1.3, about 1.35, about 1.4, about 1.45, about 1.5,about 1.55, about 1.6, about 1.65, about 1.7, about 1.75, about 1.8,about 1.85, about 1.9, about 1.95, or about 2.0 mg/kg, based on thetotal weight of siRNA (in mg) per kg of body weight.

The dosage can be about 0.1, about 0.2, about 0.3, about 0.4, about 0.7,about 1.0, about 1.25, about 1.5, about 1.7, about 2.0, about 3.0, orabout 6.0 mg/kg.

The dosage can be about 0.1, about 0.2, about 0.3, about 0.4, about 0.7,about 1.25, about 1.5, about 1.7, or about 6.0 mg/kg.

The dosage can be about 0.1, about 0.2, about 0.4, about 0.7, about 1.0,about 1.25, about 1.50 or about 1.70 mg/kg.

The dosage can be about 0.1, about 0.2, about 0.4, about 0.7, about 1.0,about 1.25, or about 1.50 mg/kg.

In one embodiment, the dosage is about 0.7, about 1.0, about 1.25 orabout 1.5 mg/kg.

In one embodiment the dosage is at least about 0.4 mg/kg. In anotherembodiment the dosage is at least about 0.7 mg/kg. In anotherembodiment, the dosage is at least about 1.0 mg/kg.

In a preferred embodiment, the dosage is about 1.0 mg/kg. In anotherpreferred embodiment, the dosage is about 1.25 mg/kg.

In some embodiments, the method includes administering the composition,e.g., ALN-VSP02, once every two weeks. The course of administration canbe 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12, weeks, 14 weeks, orlonger. The patient can receive 1-20, or 1-10, or 1-5 doses. the patientcan receive 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses.

In one embodiment, the composition is administered in a single doseevery other week for four weeks for a total of two doses. In anotherembodiment, the composition is administered in a single dose every otherweek for eight weeks for a total of four doses.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Administration

The compositions of the present invention may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. Administration may be topical,pulmonary, e.g., by inhalation or insufflation of powders or aerosols,including by nebulizer; intratracheal, intranasal, epidermal andtransdermal, and subdermal, oral or parenteral, e.g., subcutaneous.

In one embodiment, the composition, e.g., ALN-VSP02, is administeredsystemically via parental means. Parenteral administration includesintravenous (IV), intra-arterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g.,intraparenchymal, intrathecal or intraventricular, administration. Forexample, ALN-VSP02 can be administered intravenously to a patient.

Intravenous infusion or injection can be administered for about 15minutes. Intravenous infusion or injection can be administered for about30 minutes. Intravenous infusion or injection can be administered forabout 60 minutes. Intravenous infusion or injection can also beadministered over the course of about 1 to about 5 minutes, about 5 toabout 10 minutes, about 10 to about 20 minutes, about 20 to about 30minutes, or longer.

In one embodiment, the infusion or injection is administered over thecourse of about 15 to about 30 minutes. In another embodiment, theinfusion or injection is administered over the course of about 30 toabout 60 minutes

In certain instances, administration of siRNA treatment via IV infusioncan cause an acute adverse reaction. Accordingly, in one embodiment ofthe invention, the duration of the IV infusion is extended if it isobserved or predicted that a patient had or will have an acute adversereaction to an siRNA treatment. In one aspect, the duration of IVinfusion is extended to more than 15, 30, 45, or 60 minutes. In anotheraspect, the duration of IV infusion is extended to more than 1, 2, 3, or4 hours. In a particular embodiment, the duration of IV infusion isextended to up to 3 hours in the event of an acute infusion reaction.For such, a dsRNA molecule can be formulated into compositions such assterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions in liquid or solid oilbases. Such solutions also can contain buffers, diluents, and othersuitable additives. For parenteral, intrathecal, or intraventricularadministration, a dsRNA molecule can be formulated into compositionssuch as sterile aqueous solutions, which also can contain buffers,diluents, and other suitable additives (e.g., penetration enhancers,carrier compounds, and other pharmaceutically acceptable carriers).Formulations are described in more detail herein.

The composition, e.g., ALN-VSP02, can be delivered in a manner to targeta particular tissue, such as the liver (e.g., the hepatocytes of theliver).

It is well known to one of skill in the art that, in certain instances,siRNA treatment can generate an off-target global or acute inflammatoryresponse, leading to possible unwanted inflammation, toxic side effects,and discomfort. Several compounds are capable of mitigating an unwantedinflammatory or pain response when provided in advance of siRNAtreatment.

Accordingly, in some embodiments of the invention, the administration ofthe composition, e.g., ALN-VSP02, is preceded by the administration ofat least one compound capable of mitigating an inflammatory response. Inone embodiment the compound selected from the group consisting ofdexamethasone, H1 and H2 blockers, and acetaminophen.

The administration of a compound for mitigating unwanted off-targeteffects can occur simultaneously to, just before, or several minutesbefore administration of an siRNA treatment. In one embodiment,administration of a compound to mitigate unwanted off-target effectsoccurs more than about 10, 15, 30, 45, or 60 minutes prior toadministration of an siRNA treatment.

Formulations

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. In one aspectare formulations that target the liver when treating hepatic disorderssuch as hyperlipidemia.

In addition, dsRNA that target the EG5/KSP and/or VEGF gene can beformulated into compositions containing the dsRNA admixed, encapsulated,conjugated, or otherwise associated with other molecules, molecularstructures, or mixtures of nucleic acids. For example, a compositioncontaining one or more dsRNA agents that target the Eg5/KSP and/or VEGFgene can contain other therapeutic agents, such as other cancertherapeutics or one or more dsRNA compounds that target non-EG5/KSPAND/OR VEGF genes.

Oral, Parenteral, Topical, and Biologic Formulations

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. dsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. dsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, U.S. Patent Publication. No. 20030027780, and U.S. Pat.No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Suitable topical formulationsinclude those in which the dsRNAs featured in the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). dsRNAs featured in the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, dsRNAs may be complexedto lipids, in particular to cationic lipids. Suitable fatty acids andesters include but are not limited to arachidonic acid, oleic acid,eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid,palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

In addition, dsRNA molecules can be administered to a mammal as biologicor abiologic means as described in, for example, U.S. Pat. No.6,271,359. Abiologic delivery can be accomplished by a variety ofmethods including, without limitation, (1) loading liposomes with adsRNA acid molecule provided herein and (2) complexing a dsRNA moleculewith lipids or liposomes to form nucleic acid-lipid or nucleicacid-liposome complexes. The liposome can be composed of cationic andneutral lipids commonly used to transfect cells in vitro. Cationiclipids can complex (e.g., charge-associate) with negatively chargednucleic acids to form liposomes. Examples of cationic liposomes include,without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP.Procedures for forming liposomes are well known in the art. Liposomecompositions can be formed, for example, from phosphatidylcholine,dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine,dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine.Numerous lipophilic agents are commercially available, includingLipofectin™ (Invitrogen/Life Technologies, Carlsbad, Calif.) andEffectene™ (Qiagen, Valencia, Calif.). In addition, systemic deliverymethods can be optimized using commercially available cationic lipidssuch as DDAB or DOTAP, each of which can be mixed with a neutral lipidsuch as DOPE or cholesterol. In some cases, liposomes such as thosedescribed by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997))can be used. In other embodiments, polycations such as polyethyleneiminecan be used to achieve delivery in vivo and ex vivo (Boletta et al., J.Am. Soc. Nephrol. 7: 1728 (1996)). Additional information regarding theuse of liposomes to deliver nucleic acids can be found in U.S. Pat. No.6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005.Nat Biotechnol, 23(8):1002-7.

Biologic delivery can be accomplished by a variety of methods including,without limitation, the use of viral vectors. For example, viral vectors(e.g., adenovirus and herpes virus vectors) can be used to deliver dsRNAmolecules to liver cells. Standard molecular biology techniques can beused to introduce one or more of the dsRNAs provided herein into one ofthe many different viral vectors previously developed to deliver nucleicacid to cells. These resulting viral vectors can be used to deliver theone or more dsRNAs to cells by, for example, infection.

Liposomal Formulations

The compositions used in the invention can be in a liposome formulation.As used in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interior.The aqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; and liposomescan protect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether)were used to deliver cyclosporin-A into the dermis of mouse skin.Results indicated that such non-ionic liposomal systems were effectivein facilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al., S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes, it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a dsRNA featured in the invention is fullyencapsulated in the lipid formulation, e.g., to form a nucleicacid-lipid particle. Nucleic acid-lipid particles typically contain acationic lipid, a non-cationic lipid, a sterol, and a lipid thatprevents aggregation of the particle (e.g., a PEG-lipid conjugate).Nucleic acid-lipid particles are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). In addition, thenucleic acids when present in the nucleic acid-lipid particles of thepresent invention are resistant in aqueous solution to degradation witha nuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

Nucleic acid-lipid particles can further include one or more additionallipids and/or other components such as cholesterol. Other lipids may beincluded in the liposome compositions for a variety of purposes, such asto prevent lipid oxidation or to attach ligands onto the liposomesurface. Any of a number of lipids may be present, includingamphipathic, neutral, cationic, and anionic lipids. Such lipids can beused alone or in combination. Specific examples of additional lipidcomponents that may be present are described herein.

Additional components that may be present in a nucleic acid-lipidparticle include bilayer stabilizing components such as polyamideoligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins,detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat.No. 5,885,613).

A nucleic acid-lipid particle can include one or more of a second aminolipid or cationic lipid, a neutral lipid, a sterol, and a lipid selectedto reduce aggregation of lipid particles during formation, which mayresult from steric stabilization of particles which preventscharge-induced aggregation during formation.

Nucleic acid-lipid particles include, e.g., a SPLP, pSPLP, and SNALP.The term“SNALP” refers to a stable nucleic acid-lipid particle,including SPLP. The term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SPLPsinclude “pSPLP,” which include an encapsulated condensing agent-nucleicacid complex as set forth in PCT Publication No. WO 00/03683.

The nucleic acid-lipid particles of the present invention typically havea mean diameter of about 50 nm to about 150 nm, more typically about 60nm to about 130 nm, more typically about 70 nm to about 110 nm, mosttypically about 70 nm to about 90 nm, or about 50, 60, 70, 80, 90, 100,110, 120, 130, 140, or about 150 nm such that the particles aresubstantially nontoxic

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1, or about 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, or 33:1.

Cationic Lipids

The nucleic acid-lipid particles of the invention typically include acationic lipid. The cationic lipid may be, for example,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALNY-100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200), or a mixture thereof.

Other cationic lipids, which carry a net positive charge at aboutphysiological pH, in addition to those specifically described above, mayalso be included in lipid particles of the invention. Such cationiclipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammoniumchloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammoniumchloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL). In particular embodiments, acationic lipid is an amino lipid.

As used herein, the term “amino lipid” is meant to include those lipidshaving one or two fatty acid or fatty alkyl chains and an amino headgroup (including an alkylamino or dialkylamino group) that may beprotonated to form a cationic lipid at physiological pH.

Other amino lipids would include those having alternative fatty acidgroups and other dialkylamino groups, including those in which the alkylsubstituents are different (e.g., N-ethyl-N-methylamino-,N-propyl-N-ethylamino- and the like). For those embodiments in which R¹¹and R¹² are both long chain alkyl or acyl groups, they can be the sameor different. In general, amino lipids having less saturated acyl chainsare more easily sized, particularly when the complexes must be sizedbelow about 0.3 microns, for purposes of filter sterilization. Aminolipids containing unsaturated fatty acids with carbon chain lengths inthe range of C₁₄ to C₂₂ are preferred. Other scaffolds can also be usedto separate the amino group and the fatty acid or fatty alkyl portion ofthe amino lipid. Suitable scaffolds are known to those of skill in theart.

In certain embodiments, amino or cationic lipids of the invention haveat least one protonatable or deprotonatable group, such that the lipidis positively charged at a pH at or below physiological pH (e.g. pH7.4), and neutral at a second pH, preferably at or above physiologicalpH. It will, of course, be understood that the addition or removal ofprotons as a function of pH is an equilibrium process, and that thereference to a charged or a neutral lipid refers to the nature of thepredominant species and does not require that all of the lipid bepresent in the charged or neutral form. Lipids that have more than oneprotonatable or deprotonatable group, or which are zwiterrionic, are notexcluded from use in the invention.

In certain embodiments, protonatable lipids according to the inventionhave a pKa of the protonatable group in the range of about 4 to about11. Most preferred is pKa of about 4 to about 7, because these lipidswill be cationic at a lower pH formulation stage, while particles willbe largely (though not completely) surface neutralized at physiologicalpH around pH 7.4. One of the benefits of this pKa is that at least somenucleic acid associated with the outside surface of the particle willlose its electrostatic interaction at physiological pH and be removed bysimple dialysis; thus greatly reducing the particle's susceptibility toclearance.

In one embodiment, the cationic lipid is1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA). Synthesis andpreparation of nucleic acid-lipid particles including DLinDMA isdescribed in International application number PCT/CA2009/00496, filedApr. 15, 2009.

In one embodiment, the cationic lipid XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1.3]-dioxolane) is used to preparenucleic acid-lipid particles. Synthesis of XTC is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In another embodiment, the cationic lipid MC3((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), (e.g., DLin-M-C3-DMA) is used to preparenucleic acid-lipid particles. Synthesis of MC3 and MC3 comprisingformulations are described, e.g., in U.S. Provisional Ser. No.61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, which are hereby incorporated byreference.

In another embodiment, the cationic lipid ALNY-100((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)is used to prepare nucleic acid-lipid particles. Synthesis of ALNY-100is described in International patent application number PCT/US09/63933filed on Nov. 10, 2009, which is herein incorporated by reference.

In another embodiment, the cationic lipid1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200) is used to prepare nucleic acid lipid particles. C12-200 isalso known as Tech G1. Synthesis of C12-200 and formulations usingC12-200 are described in International patent application no.PCT/US10/33777 filed May 5, 2010 and in Love et al (Love et al. (2010)PNAS 107(5); 1864-69).

The cationic lipid, e.g., DLinDMA, can comprise from about 20 mol % toabout 70 mol % or about 45-65 mol % or about 20, 25, 30, 35, 40, 45, 50,55, 60, 65, or about 70 mol % of the total lipid present in theparticle. In one embodiment the cationic lipid comprises about 57.1 mol% of the total lipid present.

Non-Cationic Lipids

The nucleic acid-lipid particles of the invention can include anon-cationic lipid. The non-cationic lipid may be an anionic lipid or aneutral lipid. Examples include but not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof.

Anionic lipids suitable for use in lipid particles of the inventioninclude, but are not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, andother anionic modifying groups joined to neutral lipids.

Neutral lipids, when present in the lipid particle, can be any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Theselection of neutral lipids for use in the particles described herein isgenerally guided by consideration of, e.g., liposome size and stabilityof the liposomes in the bloodstream. Preferably, the neutral lipidcomponent is a lipid having two acyl groups, (i.e.,diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipidshaving a variety of acyl chain groups of varying chain length and degreeof saturation are available or may be isolated or synthesized bywell-known techniques. In one group of embodiments, lipids containingsaturated fatty acids with carbon chain lengths in the range of C₁₄ toC₂₂ are preferred. In another group of embodiments, lipids with mono- ordi-unsaturated fatty acids with carbon chain lengths in the range of C₁₄to C₂₂ are used. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. Preferably, the neutrallipids used in the invention are DOPE, DSPC, POPC, or any relatedphosphatidylcholine. The neutral lipids useful in the invention may alsobe composed of sphingomyelin, dihydrosphingomyeline, or phospholipidswith other head groups, such as serine and inositol.

In one embodiment the non-cationic lipid isdistearoylphosphatidylcholine (DSPC). In another embodiment thenon-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).

The non-cationic lipid, e.g., DPPC, can be from about 5 mol % to about90 mol %, about 5 mol % to about 10 mol %, about 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90 mol % of thetotal lipid present in the particle. In one embodiment, the cationiclipid, e.g., DPPC, comprises 7.1 mol % of the nucleic acid-lipidparticle.

Conjugated Lipids

Conjugated lipids can be used in nucleic acid-lipid particle to preventaggregation, including polyethylene glycol (PEG)-modified lipids,monosialoganglioside Gm1, and polyamide oligomers (“PAO”) such as(described in U.S. Pat. No. 6,320,017). Other compounds with uncharged,hydrophilic, steric-barrier moieties, which prevent aggregation duringformulation, like PEG, Gm1 or ATTA, can also be coupled to lipids foruse as in the methods and compositions of the invention. ATTA-lipids aredescribed, e.g., in U.S. Pat. No. 6,320,017, and PEG-lipid conjugatesare described, e.g., in U.S. Pat. Nos. 5,820,873, 5,534,499 and5,885,613. Typically, the concentration of the lipid component selectedto reduce aggregation is about 1 to 15% (by mole percent of lipids).

Specific examples of PEG-modified lipids (or lipid-polyoxyethyleneconjugates) that are useful in the invention can have a variety of“anchoring” lipid portions to secure the PEG portion to the surface ofthe lipid vesicle. Examples of suitable PEG-modified lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which aredescribed in co-pending US Ser. No. 08/486,214, incorporated herein byreference, PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modifieddiacylglycerols and dialkylglycerols.

In embodiments where a sterically-large moiety such as PEG or ATTA areconjugated to a lipid anchor, the selection of the lipid anchor dependson what type of association the conjugate is to have with the lipidparticle. It is well known that mePEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remainassociated with a liposome until the particle is cleared from thecirculation, possibly a matter of days. Other conjugates, such asPEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidlyexchanges out of the formulation upon exposure to serum, with a T_(1/2)less than 60 mins. in some assays. As illustrated in U.S. patentapplication Ser. No. 08/486,214, at least three characteristicsinfluence the rate of exchange: length of acyl chain, saturation of acylchain, and size of the steric-barrier head group. Compounds havingsuitable variations of these features may be useful for the invention.For some therapeutic applications, it may be preferable for thePEG-modified lipid to be rapidly lost from the nucleic acid-lipidparticle in vivo and hence the PEG-modified lipid will possessrelatively short lipid anchors. In other therapeutic applications, itmay be preferable for the nucleic acid-lipid particle to exhibit alonger plasma circulation lifetime and hence the PEG-modified lipid willpossess relatively longer lipid anchors. Exemplary lipid anchors includethose having lengths of from about C₁₄ to about C₂₂, preferably fromabout C₁₄ to about C₁₆. In some embodiments, a PEG moiety, for examplean mPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or20,000 daltons.

It should be noted that aggregation preventing compounds do notnecessarily require lipid conjugation to function properly. Free PEG orfree ATTA in solution may be sufficient to prevent aggregation. If theparticles are stable after formulation, the PEG or ATTA can be dialyzedaway before administration to a subject.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (Ci₈). Additional conjugated lipids includepolyethylene glycol-didimyristoyl glycerol (C14-PEG or PEG-C14, wherePEG has an average molecular weight of 2000 Da) (PEG-DMG);(R)-2,3-bis(octadecyloxy)propyl 1-(methoxy poly(ethyleneglycol)2000)propylcarbamate) (PEG-DSG);PEG-carbamoyl-1,2-dimyristyloxypropylamine, in which PEG has an averagemolecular weight of 2000 Da (PEG-cDMA);N-Acetylgalactosamine-((R)-2,3-bis(octadecyloxy)propyl 1-(methoxypoly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); andpolyethylene glycol-dipalmitoylglycerol (PEG-DPG).

In one embodiment the conjugated lipid is PEG-DMG or PEG-DSG. In anotherembodiment the conjugated lipid is PEG-cDMA. In still another embodimentthe conjugated lipid is PEG-DPG. Alternatively the conjugated lipid isGalNAc-PEG-DSG.

In some embodiments the conjugated lipid that prevents aggregation ofparticles is from 0 mol % to about 20 mol % or about 0.5 to about 5.0mol % or about or about 1.5 mol % or about 2.0 mol % of the total lipidpresent in the particle. The conjugated lipid can be about 0.5, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, or about 5.0 mol % of the total lipid present in the particle. Inone embodiment, the conjugated lipid, e.g., PEG-cDMA, is 1.4 mol %.

The sterol component of the lipid mixture, when present, can be any ofthose sterols conventionally used in the field of liposome, lipidvesicle or lipid particle preparation. A preferred sterol ischolesterol.

In some embodiments, the nucleic acid-lipid particle further includes asterol, e.g., cholesterol. The sterol can be about 10 to about 60 mol %or about 25 to about 40 mol % of the nucleic acid-lipid particle. Insome embodiment the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, or about 60 mol % of the total lipid present in the article. Thesterol, e.g., cholesterol, can be about 34.3 or 34.4 mol % of the totallipid in the particle.

Lipoproteins

In one embodiment, the formulations of the invention further comprise anapolipoprotein. As used herein, the tem “apolipoprotein” or“lipoprotein” refers to apolipoproteins known to those of skill in theart and variants and fragments thereof and to apolipoprotein agonists,analogues or fragments thereof described below.

Suitable apolipoproteins include, but are not limited to, ApoA-I,ApoA-II, ApoA-IV, ApoA-V and ApoE, and active polymorphic forms,isoforms, variants and mutants as well as fragments or truncated formsthereof. In certain embodiments, the apolipoprotein is a thiolcontaining apolipoprotein. “Thiol containing apolipoprotein” refers toan apolipoprotein, variant, fragment or isoform that contains at leastone cysteine residue. The most common thiol containing apolipoproteinsare ApoA-I Milano (ApoA-I_(M)) and ApoA-I Paris (ApoA-I_(P)) whichcontain one cysteine residue (Jia et al., 2002, Biochem. Biophys. Res.Comm 297: 206-13; Bielicki and Oda, 2002, Biochemistry 41: 2089-96).ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins.Isolated ApoE and/or active fragments and polypeptide analogues thereof,including recombinantly produced forms thereof, are described in U.S.Pat. Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189;5,168,045; 5,116,739; the disclosures of which are herein incorporatedby reference. ApoE3 is disclosed in Weisgraber, et al., “Human Eapoprotein heterogeneity: cysteine-arginine interchanges in the aminoacid sequence of the apo-E isoforms,” J. Biol. Chem. (1981) 256:9077-9083; and Rall, et al., “Structural basis for receptor bindingheterogeneity of apolipoprotein E from type III hyperlipoproteinemicsubjects,” Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. (See also GenBankaccession number K00396).

In certain embodiments, the apolipoprotein can be in its mature form, inits preproapolipoprotein form or in its proapolipoprotein form. Homo-and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger etal., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-IMilano (Klon et al., 2000, Biophys. J. 79:(3)1679-87; Franceschini etal., 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al.,1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol.Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.258(14):8993-9000) can also be utilized within the scope of theinvention.

In certain embodiments, the apolipoprotein can be a fragment, variant orisoform of the apolipoprotein. The term “fragment” refers to anyapolipoprotein having an amino acid sequence shorter than that of anative apolipoprotein and which fragment retains the activity of nativeapolipoprotein, including lipid binding properties. By “variant” ismeant substitutions or alterations in the amino acid sequences of theapolipoprotein, which substitutions or alterations, e.g., additions anddeletions of amino acid residues, do not abolish the activity of nativeapolipoprotein, including lipid binding properties. Thus, a variant cancomprise a protein or peptide having a substantially identical aminoacid sequence to a native apolipoprotein provided herein in which one ormore amino acid residues have been conservatively substituted withchemically similar amino acids. Examples of conservative substitutionsinclude the substitution of at least one hydrophobic residue such asisoleucine, valine, leucine or methionine for another. Likewise, thepresent invention contemplates, for example, the substitution of atleast one hydrophilic residue such as, for example, between arginine andlysine, between glutamine and asparagine, and between glycine and serine(see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term“isoform” refers to a protein having the same, greater or partialfunction and similar, identical or partial sequence, and may or may notbe the product of the same gene and usually tissue specific (seeWeisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J.Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem.260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon etal., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol.18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90;Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov etal., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985,J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem.255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sacre etal., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003, Biophys.Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.277(33):29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23):14888-93 andU.S. Pat. No. 6,372,886).

In certain embodiments, the methods and compositions of the presentinvention include the use of a chimeric construction of anapolipoprotein. For example, a chimeric construction of anapolipoprotein can be comprised of an apolipoprotein domain with highlipid binding capacity associated with an apolipoprotein domaincontaining ischemia reperfusion protective properties. A chimericconstruction of an apolipoprotein can be a construction that includesseparate regions within an apolipoprotein (i.e., homologousconstruction) or a chimeric construction can be a construction thatincludes separate regions between different apolipoproteins (i.e.,heterologous constructions). Compositions comprising a chimericconstruction can also include segments that are apolipoprotein variantsor segments designed to have a specific character (e.g., lipid binding,receptor binding, enzymatic, enzyme activating, antioxidant orreduction-oxidation property) (see Weisgraber 1990, J. Lipid Res.31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35;Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al., 1986, J.Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem.259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al.,1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al.,1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, DrugMetab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem.275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71;Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sorenson et al., 1999,Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996,Arterioscler. Throb. Vase. Biol. 16(2):328-38: Thurberg et al., J. Biol.Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill1998, J. Biol. Chem. 273(47):30979-84).

Apolipoproteins utilized in the invention also include recombinant,synthetic, semi-synthetic or purified apolipoproteins. Methods forobtaining apolipoproteins or equivalents thereof, utilized by theinvention are well-known in the art. For example, apolipoproteins can beseparated from plasma or natural products by, for example, densitygradient centrifugation or immunoaffinity chromatography, or producedsynthetically, semi-synthetically or using recombinant DNA techniquesknown to those of the art (see, e.g., Mulugeta et al., 1998, J.Chromatogr. 798(1-2): 83-90; Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson,et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528,5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 andWO 87/02062).

Apolipoproteins utilized in the invention further include apolipoproteinagonists such as peptides and peptide analogues that mimic the activityof ApoA-I, ApoA-I Milano (ApoA-I_(M)), ApoA-I Paris (ApoA-I_(P)),ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be anyof those described in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166,and 5,840,688, the contents of which are incorporated herein byreference in their entireties.

Apolipoprotein agonist peptides or peptide analogues can be synthesizedor manufactured using any technique for peptide synthesis known in theart including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166. For example, the peptides may beprepared using the solid-phase synthetic technique initially describedby Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptidesynthesis techniques may be found in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques can be found in Stuart and Young, Solid PhasePeptide. Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).Peptides may also be synthesized by solution methods as described in TheProteins, Vol. II, 3d Ed., Neurath et al., Eds., p. 105-237, AcademicPress, New York, N.Y. (1976). Appropriate protective groups for use indifferent peptide syntheses are described in the above-mentioned textsas well as in McOmie, Protective Groups in Organic Chemistry, PlenumPress, New York, N.Y. (1973). The peptides of the present inventionmight also be prepared by chemical or enzymatic cleavage from largerportions of, for example, apolipoprotein A-I.

In certain embodiments, the apolipoprotein can be a mixture ofapolipoproteins. In one embodiment, the apolipoprotein can be ahomogeneous mixture, that is, a single type of apolipoprotein. Inanother embodiment, the apolipoprotein can be a heterogeneous mixture ofapolipoproteins, that is, a mixture of two or more differentapolipoproteins. Embodiments of heterogenous mixtures of apolipoproteinscan comprise, for example, a mixture of an apolipoprotein from an animalsource and an apolipoprotein from a semi-synthetic source. In certainembodiments, a heterogenous mixture can comprise, for example, a mixtureof ApoA-I and ApoA-I Milano. In certain embodiments, a heterogeneousmixture can comprise, for example, a mixture of ApoA-I Milano and ApoA-IParis. Suitable mixtures for use in the methods and compositions of theinvention will be apparent to one of skill in the art.

If the apolipoprotein is obtained from natural sources, it can beobtained from a plant or animal source. If the apolipoprotein isobtained from an animal source, the apolipoprotein can be from anyspecies. In certain embodiments, the apolipoprotien can be obtained froman animal source. In certain embodiments, the apolipoprotein can beobtained from a human source. In preferred embodiments of the invention,the apolipoprotein is derived from the same species as the individual towhich the apolipoprotein is administered.

Other Components

In numerous embodiments, amphipathic lipids are included in lipidparticles of the invention. “Amphipathic lipids” refer to any suitablematerial, wherein the hydrophobic portion of the lipid material orientsinto a hydrophobic phase, while the hydrophilic portion orients towardthe aqueous phase. Such compounds include, but are not limited to,phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatdylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, or dilinoleylphosphatidylcholine. Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and β-acyloxyacids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols.

Also suitable for inclusion in the lipid particles of the invention areprogrammable fusion lipids. Such lipid particles have little tendency tofuse with cell membranes and deliver their payload until a given signalevent occurs. This allows the lipid particle to distribute more evenlyafter injection into an organism or disease site before it starts fusingwith cells. The signal event can be, for example, a change in pH,temperature, ionic environment, or time. In the latter case, a fusiondelaying or “cloaking” component, such as an ATTA-lipid conjugate or aPEG-lipid conjugate, can simply exchange out of the lipid particlemembrane over time. Exemplary lipid anchors include those having lengthsof from about C₁₄ to about C₂₂, preferably from about C₁₄ to about C₁₆.In some embodiments, a PEG moiety, for example an mPEG-NH₂, has a sizeof about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.

A lipid particle conjugated to a nucleic acid agent can also include atargeting moiety, e.g., a targeting moiety that is specific to a celltype or tissue. Targeting of lipid particles using a variety oftargeting moieties, such as ligands, cell surface receptors,glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies,has been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044). The targeting moieties can include the entire protein orfragments thereof. Targeting mechanisms generally require that thetargeting agents be positioned on the surface of the lipid particle insuch a manner that the targeting moiety is available for interactionwith the target, for example, a cell surface receptor. A variety ofdifferent targeting agents and methods are known and available in theart, including those described, e.g., in Sapra, P. and Allen, T M, Prog.Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res.12:1-3, (2002).

The use of lipid particles, i.e., liposomes, with a surface coating ofhydrophilic polymer chains, such as polyethylene glycol (PEG) chains,for targeting has been proposed (Allen, et al., Biochimica et BiophysicaActa 1237: 99-108 (1995); DeFrees, et al., Journal of the AmericanChemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica etBiophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal ofLiposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic andMartin, Eds) CRC Press, Boca Raton Fla. (1995). In one approach, aligand, such as an antibody, for targeting the lipid particle is linkedto the polar head group of lipids forming the lipid particle. In anotherapproach, the targeting ligand is attached to the distal ends of the PEGchains forming the hydrophilic polymer coating (Klibanov, et al.,Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBSLetters 388: 115-118 (1996)).

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.Examples of targeting moieties can also include other proteins, specificto cellular components, including antigens associated with neoplasms ortumors. Proteins used as targeting moieties can be attached to theliposomes via covalent bonds (see, Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

Production of Nucleic Acid-Lipid Particles

In one embodiment, the nucleic acid-lipid particle formulations of theinvention are produced via an extrusion method or an in-line mixingmethod.

The extrusion method (also referred to as preformed method or batchprocess) is a method where the empty liposomes (i.e. no nucleic acid)are prepared first, followed by the addition of nucleic acid to theempty liposome. Extrusion of liposome compositions through a small-porepolycarbonate membrane or an asymmetric ceramic membrane results in arelatively well-defined size distribution. Typically, the suspension iscycled through the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing. Thesemethods are disclosed in the U.S. Pat. No. 5,008,050; U.S. Pat. No.4,927,637; U.S. Pat. No. 4,737,323; Biochim Biophys Acta. 1979 Oct. 19;557(1):9-23; Biochim Biophys Acta. 1980 Oct. 2; 601(3):559-7; BiochimBiophys Acta. 1986 Jun. 13; 858(1):161-8; and Biochim. Biophys. Acta1985 812, 55-65, which are hereby incorporated by reference in theirentirety.

The in-line mixing method is a method wherein both the lipids and thenucleic acid are added in parallel into a mixing chamber. The mixingchamber can be a simple T-connector or any other mixing chamber that isknown to one skill in the art. These methods are disclosed in U.S. Pat.No. 6,534,018 and U.S. Pat. No. 6,855,277; US publication 2007/0042031and Pharmaceuticals Research. Vol. 22, No. 3, March 2005, p. 362-372,which are hereby incorporated by reference in their entirety.

It is further understood that the formulations of the invention can beprepared by any methods known to one of ordinary skill in the art.

Characterization of Nucleic Acid-Lipid Particles

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalsiRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated siRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g. 0.5% Triton-X100. The total siRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” siRNA content (as measured by thesignal in the absence of surfactant) from the total siRNA content.Percent entrapped siRNA is typically >85%. In one embodiment, theformulations of the invention are entrapped by at least 75%, at least80% or at least 90%.

For nucleic acid-lipid particle formulations, the particle size is atleast 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm,and at least 120 nm. The suitable range is typically about at least 50nm to about at least 110 nm, about at least 60 nm to about at least 100nm, or about at least 80 nm to about at least 90 nm.

LNP01

One example of synthesis of a nucleic acid-lipid particle is as follows.Nucleic acid-lipid particles are synthesized using the lipidoidND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids). This nucleic acid-lipid particleis sometimes referred to as a LNP01 particles. Stock solutions of eachin ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, andPEG-Ceramide C16 stock solutions can then be combined in a, e.g.,42:48:10 molar ratio. The combined lipid solution can be mixed withaqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanolconcentration is about 35-45% and the final sodium acetate concentrationis about 100-300 mM. Lipid-siRNA nanoparticles typically formspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Exemplary Nucleic Acid-Lipid Particle Formulations

Additional exemplary nucleic acid-lipid particle formulations aredescribed in the following table. It is to be understood that the nameof the nucleic acid-lipid particle in the table is not meant to belimiting. For example, as used herein, the term SNALP refers toformulations that include the cationic lipid DLinDMA.

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate mol %ratio Name Lipid:siRNA ratio SNALP DLinDMA/DPPC/Cholesterol/PEG-cDMA(57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 LNP-S-XXTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP10ALNY-100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP11MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP12C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP13XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~33:1 LNP14MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5 lipid:siRNA ~11:1 LNP15MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 lipid:siRNA~11:1 LNP16 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~7:1LNP17 MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 lipid:siRNA ~10:1LNP18 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~12:1LNP19 MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 lipid:siRNA ~8:1 LNP20MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP21C12-200/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 lipid:siRNA ~7:1 LNP22XTC/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 lipid:siRNA ~10:1

DLinDMA comprising formulations such as that used in ALN-VSP02 aredescribed, e.g., in application number PCT/CA2009/00496, filed Apr. 15,2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/239,686, filed Sep. 3, 2009, which is hereby incorporated byreference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, which are hereby incorporated byreference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in International patentapplication number PCT/US10/33777 filed May 5, 2010 and in Love et al(Love et al. (2010) PNAS 107(5); 1864-69) which are hereby incorporatedby reference.

Additional Formulations

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 2, p. 335; Higuchi et al., in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions areoften biphasic systems comprising two immiscible liquid phasesintimately mixed and dispersed with each other. In general, emulsionsmay be of either the water-in-oil (w/o) or the oil-in-water (o/w)variety. When an aqueous phase is finely divided into and dispersed asminute droplets into a bulk oily phase, the resulting composition iscalled a water-in-oil (w/o) emulsion. Alternatively, when an oily phaseis finely divided into and dispersed as minute droplets into a bulkaqueous phase, the resulting composition is called an oil-in-water (o/w)emulsion. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions as needed. Pharmaceuticalemulsions may also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil-in-water-in-oil(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, non-swelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988. Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3).Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Suitable bile salts include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

dsRNAs of the present invention can be formulated in a pharmaceuticallyacceptable carrier or diluent. A “pharmaceutically acceptable carrier”(also referred to herein as an “excipient”) is a pharmaceuticallyacceptable solvent, suspending agent, or any other pharmacologicallyinert vehicle. Pharmaceutically acceptable carriers can be liquid orsolid, and can be selected with the planned manner of administration inmind so as to provide for the desired bulk, consistency, and otherpertinent transport and chemical properties. Typical pharmaceuticallyacceptable carriers include, by way of example and not limitation:water; saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extra-circulatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it isco-administered with polyinosinic acid, dextran sulfate, polycytidicacid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyaoet al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA &Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Combination Therapy

In one aspect, a composition, e.g., ALN-VSP02, of the invention can beused in combination therapy. The term “combination therapy” includes theadministration of the subject compounds in further combination withother biologically active ingredients (such as, but not limited to, asecond and different antineoplastic agent) and non-drug therapies (suchas, but not limited to, surgery or radiation treatment). For instance,the compounds of the invention can be used in combination with otherpharmaceutically active compounds, preferably compounds that are able toenhance the effect of the compounds of the invention. The compounds ofthe invention can be administered simultaneously (as a singlepreparation or separate preparation) or sequentially to the other drugtherapy. In general, a combination therapy envisions administration oftwo or more drugs during a single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFα-R, PDGIβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more agents that modulatenon-kinase biological targets or processes. Such targets include histonedeacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins(e.g., HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplasticagents (e.g. small molecules, monoclonal antibodies, antisense RNA, andfusion proteins) that inhibit one or more biological targets such asZolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar,Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin,Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 andAEE788. Such combinations may enhance therapeutic efficacy over efficacyachieved by any of the agents alone and may prevent or delay theappearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives (Mechlorethamine,cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines(thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazinesand Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide),Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide andmetal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloidssuch as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxeland Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine andVinorelbine), and Camptothecan analogs (Irinotecan and Topotecan);anti-tumor antibiotics such as Chromomycins (Dactinomycin andPlicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin,Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibioticssuch as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such asfolic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed,Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine,Cytarabine, Capecitabine, and Gemcitabine), purine antagonists(6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors(Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine,Nelarabine and Pentostatin); topoisomerase inhibitors such astopoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase IIinhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide);monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab,Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); and retinoids (Bexarotene, Isotretinoin,Tretinoin (ATRA). In certain preferred embodiments, the compounds of theinvention are administered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al. (1995) J.Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr.et al. claim a method for treating a resectable carcinoma to preventrecurrence or metastases, comprising surgically removing the tumor,dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude steroids, such as corticosteroids (including, but not limitedto, amcinonide, betamethasone, betamethasone dipropionate, betamethasonevalerate, budesonide, clobetasol, clobetasol acetate, clobetasolbutyrate, clobetasol 17-propionate, cortisone, deflazacort,desoximetasone, diflucortolone valerate, dexamethasone, dexamethasonesodium phosphate, desonide, furoate, fluocinonide, fluocinoloneacetonide, halcinonide, hydrocortisone, hydrocortisone butyrate,hydrocortisone sodium succinate, hydrocortisone valerate, methylprednisolone, mometasone, prednicarbate, prednisolone, triamcinolone,triamcinolone acetonide, and halobetasol proprionate); a 5HTi agonist,such as a triptan (e.g. sumatriptan or naratriptan); an adenosine A1agonist; an EP ligand; an NMDA modulator, such as a glycine antagonist;a sodium channel blocker (e.g. lamotrigine); a substance P antagonist(e.g. an NKi antagonist); a cannabinoid; acetaminophen or phenacetin; a5-lipoxygenase inhibitor; a leukotriene receptor antagonist; a DMARD(e.g. methotrexate); gabapentin and related compounds; a tricyclicantidepressant (e.g. amitryptilline); a neurone stabilizingantiepileptic drug; a mono-aminergic uptake inhibitor (e.g.venlafaxine); a matrix metalloproteinase inhibitor; a nitric oxidesynthase (NOS) inhibitor, such as an iNOS or an nNOS inhibitor; aninhibitor of the release, or action, of tumour necrosis factor α; anantibody therapy, such as a monoclonal antibody therapy; an antiviralagent, such as a nucleoside inhibitor (e.g. lamivudine) or an immunesystem modulator (e.g. interferon); an opioid analgesic; a localanaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g.ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g.aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); adecongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine,oxymetazoline, epinephrine, naphazoline, xylometazoline,propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine,hydrocodone, carmiphen, carbetapentane, or dextramethorphan); adiuretic; or a sedating or non-sedating antihistamine.

In one embodiment, the compositions of the invention are administered incombination with a corticosteroid. For example, a corticosteroid may beadministered just prior to the administration of a composition of theinvention (such as ALN-VSP02). In one embodiment, a corticosteroid isorally administered the night before the siRNA is administered, and acorticosteroid is administered intravenously the same day as the siRNA.

The compounds of the invention can be co-administered with siRNA thattarget other genes. For example, a compound of the invention can beco-administered with an siRNA targeted to a c-Myc gene. In one example,AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myctargeted siRNAs are disclosed in U.S. patent application Ser. No.12/373,039 which is herein incorporated by reference.

Pharmacokinetics

The invention relates in particular to a method of treating a subject inneed of treatment, where a composition, such as ALN-VSP02, isadministered to the subject, resulting in a measurable C_(max) and AUCin the plasma of the subject. The invention also relates to acomposition, such as ALN-VSP02, used to treat a subject in need oftreatment, where administration of the composition, such as ALN-VSP02,to the subject results in a measurable C_(max) and AUC in the plasma ofthe subject. The C_(max) and AUC are measurable for both VEGF and KSPsiRNA components of composition, such as ALN-VSP02, e.g, for the siRNAsAD-3133 and AD-12115.

C_(max) is defined as the peak plasma concentration of a drug afteradministration of a dosage. It is an indication of the bioavailabilityand rate of absorption of a composition or drug into the plasma of thebloodstream of a subject following administration of the composition toa subject. As one of skill in the art will realize, C_(max) isdetermined by taking multiple samples of the patient's blood atdifferent time points after administration of a composition to asubject, and measuring the plasma concentration of the composition ineach sample. In one embodiment of the present invention, C_(max) islinearly correlated with dosage concentration. In one aspect, the meanC_(max) for VEGF or KSP siRNA in the subject's plasma is between 0.4 to13 μg/mL, the C_(max) range linearly correlating with an intravenouslyadministered dosage in the range of 0.1 to 0.7 mg/kg. In certaininstances, mean C_(max) for VEGF or KSP siRNA is in the range of about0.4 to 1 μg/mL, 1.8 to 3 μg/mL, 2 to 5 μg/mL, or 5 to 14 μg/mL. In otherinstances mean C_(max) values for VEGF or KSP siRNA are greater than 13μg/mL.

In another aspect, the mean C_(max) for KSP siRNA in the subject'splasma is 0.4 to 13 μg/mL, and/or the mean C_(max) for VEGF siRNA in thesubject's plasma is 0.4 to 18 μg/mL, the C_(max) range linearlycorrelating with an intravenously administered dosage in the range of0.1 to 1.0 mg/kg. In certain instances, mean C_(max) for KSP siRNA is inthe range of about 4.0 to 10.0 μg/mL. In other instances, mean C_(max)values for VEGF siRNA is in the range of 10 to 18 μg/mL.

The Tables and Examples below provide values for mean C_(max) values andranges at various doses of the ALN-VSP02 composition.

AUC refers to the area under the curve of the concentration of a drug orcomposition in the plasma of the bloodstream over time after a dose isadministered to a patient. It is affected by the rate of absorption intoand the rate of removal of the drug or composition from the patient'sblood plasma. As one of skill in the art knows, AUC can be determined bycalculating the integral of the plasma composition concentration afterthe composition is administered. In another aspect, AUC can be predictedusing the following formula:Predicted AUC=(D×F)/CL

where D is the dosage concentration, F is a measure of bioavailability,and CL is the predicted rate of clearance. In one embodiment of theinvention, F is approximately 1 for intravenous dosage and predictedmean CL is about 1.21 ml/(min*kg) in an average human. One of skill inthe art appreciates that the values for the predicted AUC have an errorin the range of ±3- to 4-fold.

In some embodiments, the data for determining AUC is obtained by takingblood samples from the patient at various time intervals afteradministration of the composition. In one aspect, the mean AUC of ansiRNA in the patient's plasma after administration of a composition,such as ALN-VSP02, is in the range of about 10 to 1450 μg*min/mL or 10to 800 μg*min/mL. In certain instances, the mean AUC of the siRNA, is inthe range of about 10 to 50 μg*min/mL, 85 to 200 μg*min/mL, 160 to 250μg*min/mL, 300 to 800 μg*min/mL, or 300 to 1450 μg*min/mL. In otherinstances, the mean AUC of the siRNA are greater than 800 μg*min/mL.

In another aspect, the mean AUC of the siRNA in the patient's plasmaafter administration of the composition, such as ALN-VSP02, is in therange of about 10 to 1100 μg*min/mL for the KSP siRNA and 10 to 1200μg*min/mL for VEGF siRNA. The Tables and Examples below provide valuesfor mean AUC values and ranges at various doses of the ALN-VSP02composition.

In one embodiment, for the first administration in a subject of acomposition, such as ALN-VSP02, at a dosage of 1.0 mg/kg, theAUC_(0-last) of KSP siRNA is within about 80% to about 125% of15393.10±6002.12 ng*hr/mL. In another embodiment, for the secondadministration in a subject of a composition, such as ALN-VSP02, at adosage of 1.0 mg/kg, the AUC_(0-last) of KSP siRNA is within about 80%to about 125% of 9984.02±2517.30 ng*hr/mL.

In one embodiment, for the first administration in a subject of acomposition, such as ALN-VSP02, at dosage of 1.0 mg/kg, the C_(max) ofKSP siRNA is within about 80% to about 125% of 15314±5347.86 ng/mL. Inanother embodiment, for the second administration in a subject of acomposition, such as ALN-VSP02, at a dosage of 1.0 mg/kg, the C_(max) ofKSP siRNA is within about 80% to about 125% of 12440.80±9615.15 ng/mL.

In one embodiment, for the first administration in a subject of acomposition, such as ALN-VSP02, at dosage of 1.0 mg/kg, the AUC_(0-last)of VEGF siRNA is within about 80% to about 125% of 16404.33±7280.14ng*hr/mL. In another embodiment, for the second administration in asubject of a composition, such as ALN-VSP02, at dosage of 1.0 mg/kg, theAUC_(0-last) of VEGF siRNA is within about 80% to about 125% of9563.98±2173.22 ng*hr/mL.

In one embodiment, for the first administration in a subject of acomposition, such as ALN-VSP02, at dosage of 1.0 mg/kg, the C_(max) ofVEGF siRNA is within about 80% to about 125% of 15843.73±7014.77 ng/mL.In another embodiment, for the second administration in a subject of acomposition, such as ALN-VSP02, at dosage of 1.0 mg/kg, the C_(max) ofVEGF siRNA is within about 80% to about 125% of 12681.94±8811.54 ng/mL.

It is understood that the plasma concentration of a composition, such asALN-VSP02, may vary significantly between subjects, due to variabilitywith respect to metabolism and/or possible interactions with othertherapeutic agents. In accordance with one aspect of the presentinvention, the blood plasma concentration of a compound, such asALN-VSP02, may vary from subject to subject. Likewise, values such asmaximum plasma concentration (C_(max)) or time to reach maximum plasmaconcentration (T_(max)) or area under the curve from time zero to timeof last measurable concentration (AUC_(last)) or total area under theplasma concentration time curve (AUC) may vary from subject to subject.Due to this variability, the amount necessary to constitute “atherapeutically effective amount” of a compound, such as ALN-VSP02, mayvary from subject to subject.

Methods for Treating Diseases Caused by Expression of the Eg5 and VEGFGenes

The invention relates in particular to the use of a composition, e.g.,ALN-VSP02 and the like, containing at least two dsRNAs, one targeting anEg5/KSP gene, and one targeting a VEGF gene, for the treatment of acancer, such as liver cancer, e.g., for inhibiting tumor growth andtumor metastasis. For example, a composition, such as pharmaceuticalcomposition, may be used for the treatment of solid tumors, likeintrahepatic tumors such as may occur in cancers of the liver.

A composition containing a dsRNA targeting Eg5/KSP and a dsRNA targetingVEGF may also be used to treat other tumors and cancers, such as breastcancer, lung cancer, head and neck cancer, brain cancer, abdominalcancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, tongue cancer, neuroblastoma,osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer,retinoblastoma, Wilm's tumor, multiple myeloma and for the treatment ofskin cancer, like melanoma, for the treatment of lymphomas and bloodcancer. The invention further relates to the use of a compositioncontaining an Eg5 dsRNA and a VEGF dsRNA for inhibiting accumulation ofascites fluid and pleural effusion in different types of cancer, e.g.,liver cancer, breast cancer, lung cancer, head cancer, neck cancer,brain cancer, abdominal cancer, colon cancer, colorectal cancer,esophagus cancer, gastrointestinal cancer, glioma, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer,melanoma, lymphomas and blood cancer. Owing to the inhibitory effects onEg5 and VEGF expression, a composition according to the invention or apharmaceutical composition prepared therefrom can enhance the quality oflife.

In one embodiment, a patient having a tumor associated with AFPexpression, or a tumor secreting AFP, e.g., a hepatoma or teratoma, istreated. In certain embodiments, the patient has a malignant teratoma,an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, ahepatoblastoma, a heptocellular carcinoma, testicular cancer or ovariancancer.

In yet another aspect, the invention provides a method for inhibitingthe expression of the Eg5 gene and the VEGF gene in a mammal. The methodincludes administering a composition featured in the invention to themammal such that expression of the target Eg5 gene and the target VEGFgene is reduced.

The invention furthermore relates to the use of a dsRNA or apharmaceutical composition thereof, e.g., ALN-VSP02, for treating canceror for preventing tumor metastasis, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating cancer and/or forpreventing tumor metastasis. Preference is given to a combination withradiation therapy and chemotherapeutic agents, such as cisplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

The invention can also be practiced by including with a specific RNAiagent, in combination with another anti-cancer chemotherapeutic agent,such as any conventional chemotherapeutic agent. The combination of aspecific binding agent with such other agents can potentiate thechemotherapeutic protocol. Numerous chemotherapeutic protocols willpresent themselves in the mind of the skilled practitioner as beingcapable of incorporation into the method of the invention. Anychemotherapeutic agent can be used, including alkylating agents,antimetabolites, hormones and antagonists, radioisotopes, as well asnatural products. For example, the compound of the invention can beadministered with antibiotics such as doxorubicin and otheranthracycline analogs, nitrogen mustards such as cyclophosphamide,pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxoland its natural and synthetic derivatives, and the like. As anotherexample, in the case of mixed tumors, such as adenocarcinoma of thebreast, where the tumors include gonadotropin-dependent andgonadotropin-independent cells, the compound can be administered inconjunction with leuprolide or goserelin (synthetic peptide analogs ofLH-RH). Other antineoplastic protocols include the use of a tetracyclinecompound with another treatment modality, e.g., surgery, radiation,etc., also referred to herein as “adjunct antineoplastic modalities.”Thus, the method of the invention can be employed with such conventionalregimens with the benefit of reducing side effects and enhancingefficacy.

When the organism to be treated is a mammal such as a human, thecomposition may be administered by any means known in the art including,but not limited to oral or parenteral routes, including intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

The compositions and methods of the invention can be used to achieve anumber of functional endpoints in addition to inhibiting expression ofVEGF and/or Eg5 (KSP). These functional endpoints include but are notlimited to extending survival, preventing tumor formation, reducingtumor formation, reducing tumor growth rate, increasing tumor cellmonoaster formation, increasing tumor cell aberrant mitotic figureformation, reducing intratumoral hemorrhage, and reducing tumormicrovessel density, e.g., when compared to a control.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

For screening of dsRNA, single-stranded RNAs were produced by solidphase synthesis on a scale of 1 mole using an Expedite 8909 synthesizer(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) andcontrolled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg,Germany) as solid support. RNA and RNA containing 2′-O-methylnucleotides were generated by solid phase synthesis employing thecorresponding phosphoramidites and 2′-O-methyl phosphoramidites,respectively (Proligo Biochemie GmbH, Hamburg, Germany). These buildingblocks were incorporated at selected sites within the sequence of theoligoribonucleotide chain using standard nucleoside phosphoramiditechemistry such as described in Current protocols in nucleic acidchemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., NewYork, N.Y., USA. Phosphorothioate linkages were introduced byreplacement of the iodine oxidizer solution with a solution of theBeaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%).Further ancillary reagents were obtained from Mallinckrodt Baker(Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

dsRNA Targeting the Eg5 Gene

Initial Screening Set

siRNA design was carried out to identify siRNAs targeting Eg5 (alsoknown as KSP, KIF11, HSKP, KNSL1 and TRIP5). The mRNA sequence to forhuman Eg5 was used, NCBI ACCESSION NM_004523.

Eg5/KSP human mRNA, ref NM_004523 (SEQ ID NO: 11)    1agcgcagcca ttggtccggc tactctgtct ctttttcaaa ttgaggcgcc gagtcgttgc   61ttagtttctg gggattcggg cggagacgag attagtgatt tggcggctcc gactggcgcg  121ggacaaacgc cacggccaga gtaccgggta gagagcgggg acgccgacct gcgtgcgtcg  181gtcctccagg ccacgccagc gcccgagagg gaccagggag actccggccc ctgtcggccg  241ccaagcccct ccgcccctca cagcgcccag gtccgcggcc gggccttgat tttttggcgg  301ggaccgtcat ggcgtcgcag ccaaattcgt ctgcgaagaa gaaagaggag aaggggaaga  361acatccaggt ggtggtgaga tgcagaccat ttaatttggc agagcggaaa gctagcgccc  421attcaatagt agaatgtgat cctgtacgaa aagaagttag tgtacgaact ggaggattgg  481ctgacaagag ctcaaggaaa acatacactt ttgatatggt gtttggagca tctactaaac  541agattgatgt ttaccgaagt gttgtttgtc caattctgga tgaagttatt atgggctata  601attgcactat ctttgcgtat ggccaaactg gcactggaaa aacttttaca atggaaggtg  661aaaggtcacc taatgaagag tatacctggg aagaggatcc cttggctggt ataattccac  721gtacccttca tcaaattttt gagaaactta ctgataatgg tactgaattt tcagtcaaag  781tgtctctgtt ggagatctat aatgaagagc tttttgatct tcttaatcca tcatctgatg  841tttctgagag actacagatg tttgatgatc cccgtaacaa gagaggagtg ataattaaag  901gtttagaaga aattacagta cacaacaagg atgaagtcta tcaaatttta gaaaaggggg  961cagcaaaaag gacaactgca gctactctga tgaatgcata ctctagtcgt tcccactcag 1021ttttctctgt tacaatacat atgaaagaaa ctacgattga tggagaagag cttgttaaaa 1081tcggaaagtt gaacttggtt gatcttgcag gaagtgaaaa cattggccgt tctggagctg 1141ttgataagag agctcgggaa gctggaaata taaatcaatc cctgttgact ttgggaaggg 1201tcattactgc ccttgtagaa agaacacctc atgttcctta tcgagaatct aaactaacta 1261gaatcctcca ggattctctt ggagggcgta caagaacatc tataattgca acaatttctc 1321ctgcatctct caatcttgag gaaactctga gtacattgga atatgctcat agagcaaaga 1381acatattgaa taagcctgaa gtgaatcaga aactcaccaa aaaagctctt attaaggagt 1441atacggagga gatagaacgt ttaaaacgag atcttgctgc agcccgtgag aaaaatggag 1501tgtatatttc tgaagaaaat tttagagtca tgagtggaaa attaactgtt caagaagagc 1561agattgtaga attgattgaa aaaattggtg ctgttgagga ggagctgaat agggttacag 1621agttgtttat ggataataaa aatgaacttg accagtgtaa atctgacctg caaaataaaa 1681cacaagaact tgaaaccact caaaaacatt tgcaagaaac taaattacaa cttgttaaag 1741aagaatatat cacatcagct ttggaaagta ctgaggagaa acttcatgat gctgccagca 1801agctgcttaa cacagttgaa gaaactacaa aagatgtatc tggtctccat tccaaactgg 1861atcgtaagaa ggcagttgac caacacaatg cagaagctca ggatattttt ggcaaaaacc 1921tgaatagtct gtttaataat atggaagaat taattaagga tggcagctca aagcaaaagg 1981ccatgctaga agtacataag accttatttg gtaatctgct gtcttccagt gtctctgcat 2041tagataccat tactacagta gcacttggat ctctcacatc tattccagaa aatgtgtcta 2101ctcatgtttc tcagattttt aatatgatac taaaagaaca atcattagca gcagaaagta 2161aaactgtact acaggaattg attaatgtac tcaagactga tcttctaagt tcactggaaa 2221tgattttatc cccaactgtg gtgtctatac tgaaaatcaa tagtcaacta aagcatattt 2281tcaagacttc attgacagtg gccgataaga tagaagatca aaaaaaggaa ctagatggct 2341ttctcagtat actgtgtaac aatctacatg aactacaaga aaataccatt tgttccttgg 2401ttgagtcaca aaagcaatgt ggaaacctaa ctgaagacct gaagacaata aagcagaccc 2461attcccagga actttgcaag ttaatgaatc tttggacaga gagattctgt gctttggagg 2521aaaagtgtga aaatatacag aaaccactta gtagtgtcca ggaaaatata cagcagaaat 2581ctaaggatat agtcaacaaa atgacttttc acagtcaaaa attttgtgct gattctgatg 2641gcttctcaca ggaactcaga aattttaacc aagaaggtac aaaattggtt gaagaatctg 2701tgaaacactc tgataaactc aatggcaacc tggaaaaaat atctcaagag actgaacaga 2761gatgtgaatc tctgaacaca agaacagttt atttttctga acagtgggta tcttccttaa 2821atgaaaggga acaggaactt cacaacttat tggaggttgt aagccaatgt tgtgaggctt 2881caagttcaga catcactgag aaatcagatg gacgtaaggc agctcatgag aaacagcata 2941acatttttct tgatcagatg actattgatg aagataaatt gatagcacaa aatctagaac 3001ttaatgaaac cataaaaatt ggtttgacta agcttaattg ctttctggaa caggatctga 3061aactggatat cccaacaggt acgacaccac agaggaaaag ttatttatac ccatcaacac 3121tggtaagaac tgaaccacgt gaacatctcc ttgatcagct gaaaaggaaa cagcctgagc 3181tgttaatgat gctaaactgt tcagaaaaca acaaagaaga gacaattccg gatgtggatg 3241tagaagaggc agttctgggg cagtatactg aagaacctct aagtcaagag ccatctgtag 3301atgctggtgt ggattgttca tcaattggcg gggttccatt tttccagcat aaaaaatcac 3361atggaaaaga caaagaaaac agaggcatta acacactgga gaggtctaaa gtggaagaaa 3421ctacagagca cttggttaca aagagcagat tacctctgcg agcccagatc aacctttaat 3481tcacttgggg gttggcaatt ttatttttaa agaaaactta aaaataaaac ctgaaacccc 3541agaacttgag ccttgtgtat agattttaaa agaatatata tatcagccgg gcgcggtggc 3601tcatgcctgt aatcccagca ctttgggagg ctgaggcggg tggattgctt gagcccagga 3661gtttgagacc agcctggcca acgtggcaaa acctcgtctc tgttaaaaat tagccgggcg 3721tggtggcaca ctcctgtaat cccagctact ggggaggctg aggcacgaga atcacttgaa 3781cccaggaagc ggggttgcag tgagccaaag gtacaccact acactccagc ctgggcaaca 3841gagcaagact cggtctcaaa aacaaaattt aaaaaagata taaggcagta ctgtaaattc 3901agttgaattt tgatatctac ccatttttct gtcatcccta tagttcactt tgtattaaat 3961tgggtttcat ttgggatttg caatgtaaat acgtatttct agttttcata taaagtagtt 4021cttttataac aaatgaaaag tatttttctt gtatattatt aagtaatgaa tatataagaa 4081ctgtactctt ctcagcttga gcttacatag gtaaatatca ccaacatctg tccttagaaa 4141ggaccatctc atgttttttt tcttgctatg acttgtgtat tttcttgcat cctccctaga 4201cttccctatt tcgctttctc ctcggctcac tttctccctt tttatttttc accaaaccat 4261ttgtagagct acaaaaggta tcctttctta ttttcagtag tcagaatttt atctagaaat 4321cttttaacac ctttttagtg gttatttcta aaatcactgt caacaataaa tctaacccta 4381gttgtatccc tcctttcagt atttttcact tgttgcccca aatgtgaaag catttcattc 4441ctttaagagg cctaactcat tcaccctgac agagttcaca aaaagcccac ttaagagtat 4501acattgctat tatgggagac cacccagaca tctgactaat ggctctgtgc ccacactcca 4561agacctgtgc cttttagaga agctcacaat gatttaagga ctgtttgaaa cttccaatta 4621tgtctataat ttatattctt ttgtttacat gatgaaactt tttgttgttg cttgtttgta 4681tataatacaa tgtgtacatg tatctttttc tcgattcaaa tcttaaccct taggactctg 4741gtatttttga tctggcaacc atatttctgg aagttgagat gtttcagctt gaagaaccaa 4801aacagaagga atatgtacaa agaataaatt ttctgctcac gatgagttta gtgtgtaaag 4861tttagagaca tctgactttg atagctaaat taaaccaaac cctattgaag aattgaatat 4921atgctacttc aagaaactaa attgatctcg tagaattatc ttaataaaat aatggctata 4981atttctctgc aaaatcagat gtcagcataa gcgatggata atacctaata aactgccctc 5041agtaaatcca tggttaataa atgtggtttc tacattaaaa aaaaaaaaaa aaaaaaaaaa 5101 a

Sequences of siRNA targeting Eg5/KSP including AD-12115 are described inU.S. patent application Ser. No. 11/694,215 filed Mar. 30, 2007 (nowU.S. Pat. No. 7,718,629) and U.S. divisional patent application Ser. No.12/754,110, filed Apr. 5, 2010 (US Patent Publication No. 2011/0015250).The contents of these applications are incorporated by reference for allpurposes. In particular, the sequences of the siRNA disclosed in theseapplications, e.g., Tables 1 and 2, are incorporated by reference forall purposes. The siRNA were synthesized and assayed for activity asdescribed.

dsRNA Targeting the VEGF Gene

Four hundred target sequences were identified within exons 1-5 of theVEGF-A121 mRNA sequence. The mRNA reference sequence was NM_003376.

human VEGF-A121 mRNA sequence. reference transcript is: NM_003376.(SEQ ID NO: 12)   1augaacuuuc ugcugucuug ggugcauugg agccuugccu ugcugcucua ccuccaccau  61gccaaguggu cccaggcugc acccauggca gaaggaggag ggcagaauca ucacgaagug 12gugaaguuca uggaugucua ucagcgcagc uacugccauc caaucgagac ccugguggac 181aucuuccagg aguacccuga ugagaucgag uacaucuuca agccauccug ugugccccug 241augcgaugcg ggggcugcug caaugacgag ggccuggagu gugugcccac ugaggagucc 301aacaucacca ugcagauuau gcggaucaaa ccucaccaag gccagcacau aggagagaug 361agcuuccuac agcacaacaa augugaaugc agaccaaaga aagauagagc aagacaagaa 421aaaugugaca agccgaggcg guga

Sequences of siRNA targeting VEGF, including AD-3133, are described inU.S. patent application Ser. No. 11/078,073 filed Mar. 11, 2005 (USPatent publication no 2006-0094032) and US continuation-in-part patentapplication Ser. No. 12/754,110, filed Jan. 25, 2006 (US patentapplication publication no. 2006-0223770). The contents of theseapplications are incorporated by reference for all purposes. Inparticular, the sequences of the siRNA disclosed in these applications,e.g., Tables 1 and 2, are incorporated by reference for all purposes.The siRNA were synthesized and assayed for activity as described.

Example 2 Eg5 siRNA In Vitro Screening Via Cell Proliferation

As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, etal [2002] Biotechniques 33: 1244-8), a cell viability assay was used forsiRNA activity screening. siRNA duplexes targeting Eg5 were tested fortheir effect on inhibition of growth of HeLa cells. Results are providedin Ser. No. 11/694,215 filed Mar. 30, 2007 (now U.S. Pat. No.7,718,629). Duplex AL-DP-6249 showed the lowest IC50 for inhibition ofcell proliferation.

Example 3 Eg5 siRNA In Vitro Screening Via mRNA Inhibition

siRNA duplexes targeting Eg5 were tested for their effect on KSP mRNAlevels in HeLA S3 cells. Results are provided in Ser. No. 11/694,215filed Mar. 30, 2007 (now U.S. Pat. No. 7,718,629). Duplex AD-12115showed a strong reduction of KSP mRNA response, having an IC20 of 0.60and 0.41 pM, an IC50 of 3.79 and 3.39 pM, and an IC80 of 23.45 and 23.45pM.

Example 4 Silencing of Liver Eg5/KSP in Juvenile Rats FollowingSingle-Bolus Administration of LNP01 Formulated siRNA

From birth until approximately 23 days of age, Eg5/KSP expression can bedetected in the growing rat liver. Target silencing with a formulatedEg5/KSP siRNA was evaluated in juvenile rats using duplex AD-6248. Thesequence of AD-6248 and results are provided in Ser. No. 11/694,215filed Mar. 30, 2007 (now U.S. Pat. No. 7,718,629).

A statistically significant reduction in liver Eg5/KSP mRNA was obtainedfollowing treatment with formulated AD6248 at a dose of 10 mg/kg.

Example 5 Silencing of Rat Liver VEGF Following Intravenous Infusion ofLNP01 Formulated VSP

A “lipidoid” formulation comprising an equimolar mixture of two siRNAswas administered to rats. As used herein, VSP refers to a compositionhaving two siRNAs, one directed to Eg5/KSP and one directed to VEGF. Forthis experiment the duplex AD3133 directed towards VEGF and AD12115directed towards Eg5/KSP were used. Since Eg5/KSP expression is nearlyundetectable in the adult rat liver, only VEGF levels were measuredfollowing siRNA treatment.

siRNA Duplexes Administered (VSP)

Duplex ID Target Sense (5′ to 3′) Antisense (5′ to 3′) AD12115 Eg5/KSPucGAGAAucuAAAcuAAcuTsT AGUuAGUUuAGAUUCUCGATsT (SEQ ID NO: 1)(SEQ ID NO: 2) AD3133 VEGF GcAcAuAGGAGAGAuGAGCUsUAAGCUcAUCUCUCCuAuGuGCusG (SEQ ID NO: 3) (SEQ ID NO: 4) Key A, G, C,U-ribonucleotides; c, u-2′-O-Me ribonucleotides; s-phosphorothioate.

Unmodified versions of each strand and the targets for each siRNA are asfollows

Eg5/KSP unmod sense 5′ UCGAGAAUCUAAACUAACUTT 3′ SEQ ID NO: 5unmod antisense 3′ TTAGUCCUUAGAUUUGAUUGA 5′ SEQ ID NO: 6 target 5′UCGAGAAUCUAAACUAACU 3′ SEQ ID NO: 7 VEGF unmod sense 5′GCACAUAGGAGAGAUGAGCUU 3′ SEQ ID NO: 8 unmod antisense 3′GUCGUGUAUCCUCUCUACUCGAA 5′ SEQ ID NO: 9 target 5′GCACAUAGGAGAGAUGAGCUU 3′ SEQ ID NO: 10Methods

Dosing of Animals.

Adult, female Sprague-Dawley rats were administered lipidoid (“LNP01”)formulated siRNA by a two-hour infusion into the femoral vein. Groups offour animals received doses of 5, 10 and 15 milligrams per kilogram(mg/kg) bodyweight of formulated siRNA. Dose level refers to the totalamount of siRNA duplex administered in the formulation. A fourth groupreceived phosphate-buffered saline Animals were sacrificed 72 hoursafter the end of the siRNA infusion. Livers were dissected, flash frozenin liquid Nitrogen and pulverized into powders.

Formulation Procedure

The lipidoid ND98.4HCl (MW 1487) (Formula 1, above), Cholesterol(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used toprepare lipid-siRNA nanoparticles. Stock solutions of each in ethanolwere prepared: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16,100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions werethen combined in a 42:48:10 molar ratio. Combined lipid solution wasmixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that thefinal ethanol concentration was 35-45% and the final sodium acetateconcentration was 100-300 mM. Lipid-siRNA nanoparticles formedspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture was in some casesextruded through a polycarbonate membrane (100 nm cut-off) using athermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In othercases, the extrusion step was omitted. Ethanol removal and simultaneousbuffer exchange was accomplished by either dialysis or tangential flowfiltration. Buffer was exchanged to phosphate buffered saline (PBS) pH7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free methodare characterized in a similar manner. Formulations are firstcharacterized by visual inspection. They should be whitish translucentsolutions free from aggregates or sediment. Particle size and particlesize distribution of lipid-nanoparticles are measured by dynamic lightscattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particlesshould be 20-300 nm, and ideally, 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA is incubated with theRNA-binding dye Ribogreen (Molecular Probes) in the presence or absenceof a formulation disrupting surfactant, 0.5% Triton-X100. The totalsiRNA in the formulation is determined by the signal from the samplecontaining the surfactant, relative to a standard curve. The entrappedfraction is determined by subtracting the “free” siRNA content (asmeasured by the signal in the absence of surfactant) from the totalsiRNA content. Percent entrapped siRNA is typically >85%. For SNALPformulation, the particle size is at least 30 nm, at least 40 nm, atleast 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90nm, at least 100 nm, at least 110 nm, and at least 120 nm. The preferredrange is about at least 50 nm to about at least 110 nm, preferably aboutat least 60 nm to about at least 100 nm, most preferably about at least80 nm to about at least 90 nm. In one example, each of the particle sizecomprises at least about 1:1 ratio of Eg5 dsRNA to VEGF dsRNA.

mRNA Measurements.

Samples of each liver powder (approximately ten milligrams) werehomogenized in tissue lysis buffer containing proteinase K. Levels ofVEGF and GAPDH mRNA were measured in triplicate for each sample usingthe Quantigene branched DNA assay (GenoSpectra). Mean values for VEGFwere normalized to mean GAPDH values for each sample. Group means weredetermined and normalized to the PBS group for each experiment.

Protein Measurements.

Samples of each liver powder (approximately 60 milligrams) werehomogenized in 1 ml RIPA buffer. Total protein concentrations weredetermined using the Micro BCA protein assay kit (Pierce). Samples oftotal protein from each animal was used to determine VEGF protein levelsusing a VEGF ELISA assay (R&D systems). Group means were determined andnormalized to the PBS group for each experiment.

Statistical Analysis.

Significance was determined by ANOVA followed by the Tukey post-hoc test

Results

Data Summary

Mean values (±standard deviation) for mRNA (VEGF/GAPDH) and protein(rel. VEGF) are shown for each treatment group. Statistical significance(p value) versus the PBS group for each experiment is shown.

TABLE 1 VEGF/GAPDH p value rel VEGF p value PBS  1.0 ± 0.17  1.0 ± 0.17 5 mg/kg 0.74 ± 0.12 <0.05 0.23 ± 0.03 <0.001 10 mg/kg 0.65 ± 0.12<0.005 0.22 ± 0.03 <0.001 15 mg/kg 0.49 ± 0.17 <0.001 0.20 ± 0.04 <0.001

Statistically significant reductions in liver VEGF mRNA and protein weremeasured at all three siRNA dose levels.

Example 6 Assessment of VSP SNALP in Mouse Models of Human HepaticTumors

These studies utilized a VSP siRNA cocktail containing dsRNAs targetingKSP/Eg5 and dsRNAs targeting VEGF. As used herein, VSP refers to acomposition having two siRNAs, one directed to Eg5/KSP and one directedto VEGF. For this experiment the duplexes AD3133 (directed towards VEGF)and AD12115 (directed towards Eg5/KSP) were used. The siRNA cocktail wasformulated in SNALPs.

The maximum study size utilized 20-25 mice. To test the efficacy of thesiRNA SNALP cocktail to treat liver cancer, 1×10^6 tumor cells wereinjected directly into the left lateral lobe of test mice. The incisionswere closed by sutures, and the mice allowed to recover for 2-5 hours.The mice were fully recovered within 48-72 hours. The SNALP siRNAtreatment was initiated 8-11 days after tumor seeding.

The SNALP formulations utilized were (i) VSP (KSP+VEGF siRNA cocktail(1:1 molar ratio)); (ii) KSP (KSP+Luc siRNA cocktail); and (iii) VEGF(VEGF+Luc siRNA cocktail). All formulations contained equal amounts (mg)of each active siRNA. All mice received a total siRNA/lipid dose, andeach cocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1%DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug usingoriginal citrate buffer conditions.

Human Hep3B Study A: Anti-Tumor Activity of VSP-SNALP

Human Hepatoma Hep3B tumors were established in scid/beige mice byintrahepatic seeding. Group A (n=6) animals were administered PBS; GroupB (n=6) animals were administered VSP SNALP; Group C (n=5) animals wereadministered KSP/Luc SNALP; and Group D (n=5) animals were administeredVEGF/Luc SNALP.

SNALP treatment was initiated eight days after tumor seeding. The SNALPwas dosed at 3 mg/kg total siRNA, twice weekly (Monday and Thursday),for a total of six doses (cumulative 18 mg/kg siRNA). The final dose wasadministered at day 25, and the terminal endpoint was at day 27.

Tumor burden was assayed by (a) body weight; (b) liver weight; (c)visual inspection+photography at day 27; (d) human-specific mRNAanalysis; and (e) blood alpha-fetoprotein levels measured at day 27.

Table 2 below illustrates the results of visual scoring of tumor burdenmeasured in the seeded (left lateral) liver lobe. Score: “−”=no visibletumor; “+”=evidence of tumor tissue at injection site; “++”=Discretetumor nodule protruding from liver lobe; “+++”=large tumor protruding onboth sides of liver lobe; “++++”=large tumor, multiple nodulesthroughout liver lobe.

TABLE 2 Mouse Tumor Burden Group A: PBS, day 27 1 ++++ 2 ++++ 3 ++ 4 +++5 ++++ 6 ++++ Group B: VSP 1 + (VEGF + KSP/Eg5, d. 27 2 − 3 − 4 − 5 ++ 6− Group C: KSP 1 + (Luc + KSP), d. 27 2 ++ 3 − 4 + 5 ++ Group D: VEGF 1++++ (Luc + VEGF), d. 27 2 − 3 ++++ 4 +++ 5 ++++

Liver weights, as percentage of body weight, are shown in FIG. 1.

Body weights are shown in FIGS. 2A-2D.

From this study, the following conclusions were made. (1) VSP SNALPdemonstrated potent anti-tumor effects in Hep3B 1H model; (2) theanti-tumor activity of the VSP cocktail appeared largely associated withthe KSP component; (3) anti-KSP activity was confirmed by single dosehistological analysis; and (4) VEGF siRNA showed no measurable effect oninhibition of tumor growth in this model.

Human Hep3B Study B: Prolonged Survival with VSP Treatment

In a second Hep3B study, human hepatoma Hep3B tumors were established byintrahepatic seeding into scid/beige mice. These mice were deficient forlymphocytes and natural killer (NK) cells, which is the minimal scopefor immune-mediated anti-tumor effects. Group A (n=6) mice wereuntreated; Group B (n=6) mice were administered luciferase (luc) 1955SNALP (Lot No. AP10-02); and Group C (n=7) mice were administered VSPSNALP (Lot No. AP10-01). SNALP was 1:57 cDMA SNALP, and 6:1 lipid:drug.

SNALP treatment was initiated eight days after tumor seeding. SNALP wasdosed at 3 mg/kg siRNA, twice weekly (Mondays and Thursdays), for atotal of six doses (cumulative 18 mg/kg siRNA). The final dose wasdelivered at day 25, and the terminal endpoint of the study was at day27.

Tumor burden was assayed by (1) body weight; (2) visualinspection+photography at day 27; (3) human-specific mRNA analysis; and(4) blood alpha-fetoprotein measured at day 27.

Body weights were measured at each day of dosing (days 8, 11, 14, 18,21, and 25) and on the day of sacrifice (FIG. 3).

TABLE 3 Tumor Burden by Mouse macroscopic observation Group A: A1R ++untreated, A1G ++++ day 27 A1W − A2R ++++ A2G +++ A2W ++++ Group B: B1R++++ 1955 Luc SNALP B1G ++++ day 27 B1W +++ B2R ++ B2G +++ B2W ++++Group C: C1R − VSP SNALP C1G − day 27 C1B − C1W + C2R + C2G + C2W −Score: “−” = no visible tumor; “+” = evidence of tumor tissue atinjection site; “++” = Discrete tumor nodule protruding from liver lobe;“+++” = large tumor protruding on both sides of liver lobe; “++++” =large tumor, multiple nodules throughout liver lobe.

The correlation between body weights and tumor burden are shown in FIGS.4, 5 and 6.

A single dose of VSP SNALP (2 mg/kg) to Hep3B mice also resulted in theformation of mitotic spindles in liver tissue samples examined byhistological staining.

Tumor burden was quantified by quantitative RT-PCR (pRT-PCR) (Taqman).Human GAPDH was normalized to mouse GAPDH via species-specific Taqmanassays. Tumor score as shown by macroscopic observation in the tableabove correlated with GADPH levels (FIG. 7A).

Serum ELISA was performed to measure alpha-fetoprotein (AFP) secreted bythe tumor. As described below, if levels of AFP go down after treatment,the tumor is not growing. Treatment with VSP lowered AFP levels in someanimals compared to treatment with controls (FIG. 7B).

Human HepB3 Study C:

In a third study, human HCC cells (HepB3) were injected directly intothe liver of SCID/beige mice, and treatment was initiated 20 days later.Group A animals were administered PBS; Group B animals were administered4 mg/kg Luc-1955 SNALP; Group C animals were administered 4 mg/kgSNALP-VSP; Group D animals were administered 2 mg/kg SNALP-VSP; andGroup E animals were administered 1 mg/kg SNALP-VSP. Treatment was witha single intravenous (iv) dose, and mice were sacrificed 24 hr. later.

Tumor burden and target silencing was assayed by qRT-PCR (Taqman). Tumorscore was also measured visually as described above, and the results areshown in the following table. hGAPDH levels, as shown in FIG. 8,correlates with macroscopic tumor score as shown in the table below.

TABLE 4 Tumor Burden by Mouse macroscopic observation Group A: PBS A2+++ A3 +++ A4 +++ Group B: 4 mg/kg B1 + Luc-1955 SNALP B2 +++ B3 +++ B4+++ Group C: 4 mg/kg C1 ++ SNALP-VSP C2 ++ C3 ++ C4 +++ Group D: 2 mg/kgD1 ++ SNALP-VSP D2 + D3 + D4 ++ Group E: 1 mg/kg E1 +++ SNALP-VSP E2 +E3 ++ E4 + Score: “+” = variable tumor take/some small tumors; “++” =Discrete tumor nodule protruding from liver lobe; “+++” = large tumorprotruding on both sides of liver lobe

Human (tumor-derived) KSP silencing was assayed by Taqman analysis andthe results are shown in FIG. 10. hKSP expression was normalized tohGAPDH. About 80% tumor KSP silencing was observed at 4 mg/kg SNALP-VSP,and efficacy was evident at 1 mg/kg. The clear bars in FIG. 9 representthe results from small (low GAPDH) tumors.

Human (tumor-derived) VEGF silencing was assayed by Taqman analysis andthe results are shown in FIG. 10. hVEGF expression was normalized tohGAPDH. About 60% tumor VEGF silencing was observed at 4 mg/kgSNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in FIG.10 represent the results from small (low GAPDH) tumors.

Mouse (liver-derived) VEGF silencing was assayed by Taqman analysis andthe results are shown in FIG. 11A. mVEGF expression was normalized tohGAPDH. About 50% liver VEGF silencing was observed at 4 mg/kgSNALP-VSP, and efficacy was evident at 1 mg/kg.

Human HepB3 Study D: Contribution of Each dsRNA to Tumor Growth

In a fourth study, human HCC cells (HepB3) were injected directly intothe liver of SCID/beige mice, and treatment was initiated 8 days later.Treatment was with intravenous (iv) bolus injections, twice weekly, fora total of six does. The final dose was administered at day 25, and theterminal endpoint was at day 27.

Tumor burden was assayed by gross histology, human-specific mRNAanalysis (hGAPDH qPCR), and blood alpha-fetoprotein levels (serum AFPvia ELISA).

In Study 1, Group A was treated with PBS, Group B was treated withSNALP-KSP+Luc (3 mg/kg), Group C was treated with SNALP-VEGF+Luc (3mg/kg), and Group D was treated with ALN-VSP02 (3 mg/kg).

In Study 2, Group A was treated with PBS; Group B was treated withSNALP-KSP+Luc (1 mg/kg), Group C was treated with ALN-VSP02 (1 mg/kg).

Both GAPDH mRNA levels and serum AFP levels were shown to decrease aftertreatment with ALN-VSP02 (FIG. 11B).

Histology Studies:

Human hepatoma Hep3B tumors were established by intrahepatic seeding inmice. SNALP treatment was initiated 20 days after tumor seeding.Tumor-bearing mice (three per group) were treated with a singleintravenous (IV) dose of (i) VSP SNALP or (ii) control (Luc) SNALP at 2mg/kg total siRNA.

Liver/tumor samples were collected for conventional H&E histology 24hours after single SNALP administration.

Large macroscopic tumor nodules (5-10 mm) were evident at necroscopy.

Effect of ALN-VSP in Hep3B mice:

ALN-VSP (a cocktail of KSP dsRNA and VEGF dsRNA) treatment reduced tumorburden and expression of tumor-derived KSP and VEGF. GAPDH mRNA levels,a measure of tumor burden, were also observed to decline followingadministration of ALN-VSP dsRNA (see FIGS. 12A-12C). A decrease in tumorburden by visual macroscopic observation was also evident followingadministration of ALN-VSP.

A single IV bolus injection of ALN-VSP also resulted in mitotic spindleformation that was clearly detected in liver tissue samples from Hep3Bmice. This observation indicated cell cycle arrest.

Example 7a Survival of SNALP-VSP Animals Versus SNALP-Luc TreatedAnimals

To test the effect of siRNA SNALP on survival rates of cancer subjects,tumors were established by intrahepatic seeding in mice and the micewere treated with SNALP-siRNA. These studies utilized a VSP siRNAcocktail containing dsRNAs targeting KSP/Eg5 and VEGF. Control was dsRNAtargeting Luc. The siRNA cocktail was formulated in SNALPs.

Tumor cells (Human Hepatoma Hep3B, 1×10^6) were injected directly intothe left lateral lobe of scid/beige mice. These mice were deficient forlymphocytes and natural killer (NK) cells, which is the minimal scopefor immune-mediated anti-tumor effects. The incisions were closed bysutures, and the mice allowed to recover for 2-5 hours. The mice werefully recovered within 48-72 hours.

All mice received a total siRNA/lipid intravenous (iv) dose, and eachcocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1%DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug usingoriginal citrate buffer conditions.

siRNA-SNALP treatment was initiated on the day indicated below (18 or 26days) after tumor seeding. siRNA-SNALP were administered twice a weekfor three weeks after 18 or 26 days at a dose of 4 mg/kg. Survival wasmonitored and animals were euthanized based on humane surrogateendpoints (e.g., animal body weight, abdominal distension/discoloration,and overall health).

The survival data for treatment initiated 18 days after tumor seeing issummarized in Table 5, Table 6, and FIG. 13A.

TABLE 5 Kaplan-Meier (survival) data (% Surviving) Day SNALP-LucSNALP-VSP 18 100% 100% 22 100% 100% 25 100% 100% 27 100% 100% 28 100%100% 28 86% 100% 29 86% 100% 32 86% 100% 33 86% 100% 33 43% 100% 35 43%100% 36 43% 100% 36 29% 100% 38 29% 100% 38 14% 100% 38 14% 88% 40 14%88% 43 14% 88% 45 14% 88% 49 14% 88% 51 14% 88% 51 14% 50% 53 14% 50% 5314% 25% 55 14% 25% 57 14% 25% 57 0% 0%

TABLE 6 Survival in days, for each animal. Treatment Animal groupSurvival 1 SNALP-Luc 28 days 2 SNALP-Luc 33 days 3 SNALP-Luc 33 days 4SNALP-Luc 33 days 5 SNALP-Luc 36 days 6 SNALP-Luc 38 days 7 SNALP-Luc 57days 8 SNALP-VSP 38 days 9 SNALP-VSP 51 days 10 SNALP-VSP 51 days 11SNALP-VSP 51 days 12 SNALP-VSP 53 days 13 SNALP-VSP 53 days 14 SNALP-VSP57 days 15 SNALP-VSP 57 days

FIG. 13A shows the mean survival of SNALP-VSP animals and SNALP-Luctreated animals versus days after tumor seeding. The mean survival ofSNALP-VSP animals was extended by approximately 15 days versus SNALP-Luctreated animals.

TABLE 7 Serum alpha fetoprotein (AFP) concentration, for each animal, ata time pre-treatment and at end of treatment (concentration in μg/ml)End of pre-Rx Rx 1 SNALP-Luc 30.858 454.454 2 SNALP-Luc 10.088 202.082 3SNALP-Luc 23.736 648.952 4 SNALP-Luc 1.696 13.308 5 SNALP-Luc 4.778338.688 6 SNALP-Luc 15.004 826.972 7 SNALP-Luc 11.036 245.01 8 SNALP-VSP37.514 182.35 9 SNALP-VSP 91.516 248.06 10 SNALP-VSP 25.448 243.13 11SNALP-VSP 24.862 45.514 12 SNALP-VSP 57.774 149.352 13 SNALP-VSP 12.44678.724 14 SNALP-VSP 2.912 9.61 15 SNALP-VSP 4.516 11.524

Tumor burden was monitored using serum AFP levels during the course ofthe experiment. Alpha-fetoprotein (AFP) is a major plasma proteinproduced by the yolk sac and the liver during fetal life. The protein isthought to be the fetal counterpart of serum albumin, and human AFP andalbumin gene are present in tandem in the same transcriptionalorientation on chromosome 4. AFP is found in monomeric as well asdimeric and trimeric forms, and binds copper, nickel, fatty acids andbilirubin. AFP levels decrease gradually after birth, reaching adultlevels by 8-12 months. Normal adult AFP levels are low, but detectable.AFP has no known function in normal adults and AFP expression in adultsis often associated with a subset of tumors such as hepatoma andteratoma. AFP is a tumor marker used to monitor testicular cancer,ovarian cancer, and malignant teratoma. Principle tumors that secreteAFP include endodermal sinus tumor (yolk sac carcinoma), neuroblastoma,hepatoblastoma, and heptocellular carcinoma. In patients withAFP-secreting tumors, serum levels of AFP often correlate with tumorsize. Serum levels are useful in assessing response to treatment.Typically, if levels of AFP go down after treatment, the tumor is notgrowing. A temporary increase in AFP immediately following chemotherapymay indicate not that the tumor is growing but rather that it isshrinking (and releasing AFP as the tumor cells die). Resection isusually associated with a fall in serum levels. As shown in FIG. 14,tumor burden in SNALP-VSP treated animals was significantly reduced.

Example 7b ALN-VSP Extends Survival

As used herein, ALN-VSP refers to lipid formulated siRNAs targeting VEGFand KSP (Eg5). The siRNAs are ALN-12115 targeting KSP and ALN-3133targeting VEGF. The lipid formulation is a SNALP formulation (describedherein) and including DLinDMA, DSPC, mPEG2000-C-DMA, and cholesterol.

Tumors were established by intrahepatic implantation of Hep3B cells asdescribed herein. 4 mg/kg of ALN-VSP or SNALP-Luc was administered twiceper week for three weeks beginning 26 days after tumor implantation.Animals were euthanized based on humane surrogate endpoints. As shown inFIG. 13B, mean survival of ALN-VSP animals was extended by approximately50% versus SNALP-Luc treated animals.

Example 8a Induction of Mono-Asters in Orthotopic HCC Model

Inhibition of KSP in dividing cells leads to the formation of monoasters that are readily observable in histological sections. Orthotopicmouse liver tumor models were used as described herein. Briefly, humanhepatoma cells (Hep3B) or human colorectal carcinoma cells (HCT116) wereinjected intrahepatically into the left lateral lobe of scid/beige mice.

To determine whether mono aster formation occurred in SNALP-VSP treatedtumors, tumor bearing animals (three weeks after Hep3B cellimplantation) were administered 2 mg/kg SNALP-VSP via tail veininjection. Control animals received 2 mg/kg SNALP-Luc. Twenty four hourslater, animals were sacrificed, and tumor bearing liver lobes wereprocessed for histological analysis. Representative images of H&Estained tissue sections are shown in FIG. 15. Extensive mono asterformation was evident in ALN VSP02 treated (FIG. 15A, but not SNALP-Luctreated FIG. 15B, tumors. In the latter, normal mitotic figures wereevident. The generation of mono asters is a characteristic feature ofKSP inhibition and provides further evidence that SNALP-VSP hassignificant activity in established liver tumors.

Example 8b Induction of Monoasters in Intraperitoneal HCC Model

A mouse HEP3B metastatic model was developed and used to assay theeffect of ALN-VSP treatment on monoaster formation in intraperitonealHEP3B tumors. HEP3B cells were obtained from ATCC and engineered tostably express Firefly Luciferase. Fox scid beige mice (8-9 weeks old)were obtained from Charles River Laboratories. 1 ml cells suspended in100 cc of sterile PBS were injected in to the peritoneal cavity. Tumorgrowth was monitored using non invasive luminescence imaging.

Animals received a single dose of ALN-VSP or SNALP-Luc at 8 mg/kg.Tumors were analyzed 48 h after dosing. Paraffin embedded sections oftumors were stained with H&E. Whole tumor sections were imaged usingfloating ROI (region of interest) analysis, and the number of simplemitoses or aberrant mitotic figures (monoasters) were counted. Totalcounts were divided by the number of ROI per tumor. As show in FIG. 20ALN-VSP treatment leads to the accumulation of aberrant mitotic figures(monoasters) in tumor tissue from intraperitoneal HCC model.

Example 8c Reduction of mRNA Levels and Induction of Monoasters inColorectal Carcinoma Tumors in Liver and Extrahepatic Sites

The efficacy of treatment with ALN-VSP in colorectal carcinoma tumorswas assayed. Tumors were established by intrahepatic implantation ofHCT116 cells directly into the livers of immunocompromised mice (i.e.,into the left lateral lobe of scid/beige mice). In some animals,disseminated tumors developed at extra-hepatic sites, including lymphnodes, lungs, and subcutaneously (s.c.). Tumor bearing animals receivedeither a single dose or multiple doses.

Tumor bearing animals received a single dose of ALN-VSP (4, 2 or 1mg/kg) or SNALP-Luc (4 mg/kg) 14 days after tumor implantation. mRNAlevels of tumor-derived (human) KSP, normalized to GAPDH, were measured24 h after drug administration using species specific TaqMan probes. Asshown in FIG. 21, ALN-VSP demonstrated 35% reduction of hKSP relative tothe SNALP-Luc control at 4 mg/kg.

Tumor bearing animals received multiple doses of ALN-VSP and SNALP-Luc14 days after tumor implantation. ALN-VSP was administered at 4.0 and1.0 mg/kg, control SNALP-Luc at 4 mg/kg twice a week for 3 weeks. Tumorbearing livers, lymph nodes, lungs and subcutaneous metastases wereanalyzed 48 h after dosing. Paraffin embedded sections of tumors werestained with H&E. Whole tumor sections were imaged using floating ROI(region of interest) analysis, and the number of simple mitoses oraberrant mitotic figures (monoasters) were counted. Total counts weredivided by the number of ROI per tumor.

As shown in FIG. 25A, FIG. 25B, and FIG. 25C, ALN-VSP treatment leads toaccumulation of aberrant mitotic figures (monoasters) in liver tumors,lung, lymph node, and subcutaneous metastases. FIGS. 22A & 25A (liver),P<0.05 (One-way ANOVA with Tukey's multiple comparison test); FIG. 25B(lung) p=0.0182 by unpaired t test as compared to monoasters inSNALP-1955 treated animals; FIGS. 22B & 25C (lymph node); FIG. 25C(subcutaneous).

The results demonstrated that each siRNA makes a distinct contributionto efficacy. ALN-VSP treatment led to accumulation of aberrant mitoticfigures (monoasters), a hallmark of KSP inhibition, in both types oforthotopic liver tumors, as well as in extra-hepatic tumors of differentorigin. Evidence of therapeutic VEGF inhibition was shown by markedreductions in tumor microvessel density and intratumoral hemorrhage inorthotopic tumors.

Example 8d ALN-VSP Reduced Intratumoral Hemorrhage and MicrovesselDensity

The effect of treatment with ALN-VSP compared to SNALP-Luc onintratumoral hemorrhage and microvessel density was analyzed in usingthe Hep3B orthotopic HCC mouse model described herein.

Two studies were performed. Study 1 compared 4 mg/kg ALN-VSP vs.SNALP-Luc administered twice per week for three weeks beginning 26 daysafter Hep3B orthotopic tumor implantation. Animals were euthanized basedon humane surrogate end points. Study 2 compared 6 mg/kg SNALP-VEGF onlyvs. SNALP-Luc administered twice per week for three weeks beginning 14days after tumor implantation. Bevacizumab at 5 mg/kg administered IPwas used as a positive control. Animals were euthanized 72 h after thelast dose.

Paraffin embedded sections of tumors were stained with H&E to revealregions of tumor hemorrhage, or with a CD34 antibody to detect tumorvasculature. Two whole tumor sections from distant tumor slabs wereimaged using floating ROI (region of interest) analysis. Regions ofintratumoral hemorrhage were outlined in H&E stained sections and totalareas of hemorrhage were quantified in each tumor. To quantifymicrovessel density, CD34 stained areas were quantified as a percentageof total tumor area.

As shown in FIG. 23, ALN-VSP treatment reduced tumor hemorrhage andmicrovessel density. As shown in FIG. 24, the vascular effects ofALN-VSP were attributable to the VEGF siRNA as SNALP-VEGF reduces tumorhemorrhage and microvessel density to the same extent as ALN-VSP.

Example 9 Manufacturing Process and Product Specification of ALN-VSP02(SNALP-VSP)

ALN-VSP02 product contains 2 mg/mL of drug substance ALN-VSPDS01formulated in a sterile lipid particle formulation (referred to asSNALP) for IV administration via infusion. Drug substance ALN-VSPDS01consists of two siRNAs (ALN-12115 targeting KSP and ALN-3133 targetingVEGF) in an equimolar ratio. The drug product is packaged in 10 mL glassvials with a fill volume of 5 mL.

The following terminology is used herein:

Drug Substance siRNA Duplexes Single Strand Intermediates ALN-VSPDS01ALN-12115* Sense: A-19562 Antisense: A-19563 ALN-3133** Sense: A-3981Antisense: A-3982 *Alternate names = AD-12115, AD12115; **Alternatenames = AD-3133, AD3133

9.1 Preparation of Drug Substance ALN-VSPDS01

The two siRNA components of drug substance ALN-VSPDS01, ALN-12115 andALN-3133, are chemically synthesized using commercially availablesynthesizers and raw materials. The manufacturing process consists ofsynthesizing the two single strand oligonucleotides of each duplex (A19562 sense and A 19563 antisense of ALN 12115 and A 3981 sense and A3982 antisense of ALN 3133) by conventional solid phase oligonucleotidesynthesis using phosphoramidite chemistry and 5′ Odimethoxytriphenylmethyl (DMT) protecting group with the 2′ hydroxylprotected with tert butyldimethylsilyl (TBDMS) or the 2′ hydroxylreplaced with a 2′ methoxy group (2′ OMe). Assembly of anoligonucleotide chain by the phosphoramidite method on a solid supportsuch as controlled pore glass or polystyrene. The cycle consists of 5′deprotection, coupling, oxidation, and capping. Each coupling reactionis carried out by activation of the appropriately protected ribo, 2′OMe, or deoxyribonucleoside amidite using 5 (ethylthio) 1H tetrazolereagent followed by the coupling of the free 5′ hydroxyl group of asupport immobilized protected nucleoside or oligonucleotide. After theappropriate number of cycles, the final 5′ protecting group is removedby acid treatment. The crude oligonucleotide is cleaved from the solidsupport by aqueous methylamine treatment with concomitant removal of thecyanoethyl protecting group as well as nucleobase protecting groups. The2′ O TBDMS group is then cleaved using a hydrogen fluoride containingreagent to yield the crude oligoribonucleotide, which is purified usingstrong anion exchange high performance liquid chromatography (HPLC)followed by desalting using ultrafiltration. The purified single strandsare analyzed to confirm the correct molecular weight, the molecularsequence, impurity profile and oligonucleotide content, prior toannealing into the duplexes. The annealed duplex intermediates ALN 12115and ALN 3133 are either lyophilized and stored at 20° C. or mixed in 1:1molar ratio and the solution is lyophilized to yield drug substance ALNVSPDS01. If the duplex intermediates were stored as dry powder, they areredissolved in water before mixing. The equimolar ratio is achieved bymonitoring the mixing process by an HPLC method.

The manufacturing process flow diagram is shown in FIG. 16.

The drug substance was assayed for storage stability (data not shown)The assay methods were chosen to assess physical property (appearance,pH, moisture), purity (by SEC and denaturing anion exchangechromatography) and potency (by denaturing anion exchange chromatography[AX-HPLC]). ALN-VSPDS01 showed stability for up to 12 months storage at20 degrees C.

9.2 Preparation of Drug Product ALN-VSP02 (SNALP-VSP)

ALN VSP02, is a sterile formulation of the two siRNAs (in a 1:1 molarratio) with lipid excipients in isotonic buffer. The lipid excipientsassociate with the two siRNAs, protect them from degradation in thecirculatory system, and aid in their delivery to the target tissue. Thespecific lipid excipients and the quantitative proportion of each (shownin Table 9) have been selected through an iterative series ofexperiments comparing the physicochemical properties, stability,pharmacodynamics, pharmacokinetics, toxicity and productmanufacturability of numerous different formulations. The excipientDLinDMA is a titratable aminolipid that is positively charged at low pH,such as that found in the endosome of mammalian cells, but relativelyuncharged at the more neutral pH of whole blood. This featurefacilitates the efficient encapsulation of the negatively charged siRNAsat low pH, preventing formation of empty particles, yet allows foradjustment (reduction) of the particle charge by replacing theformulation buffer with a more neutral storage buffer prior to use.Cholesterol and the neutral lipid DPPC are incorporated in order toprovide physicochemical stability to the particles. Thepolyethyleneglycol lipid conjugate PEG2000 C DMA aids drug productstability, and provides optimum circulation time for the proposed use.ALN VSP02 lipid particles have a mean diameter of approximately 80-90 nmwith low polydispersity values. A representative cryo transmissionelectron microscope (cryo TEM) image is shown in FIG. 17. At neutral pH,the particles are essentially uncharged, with Zeta Potential values ofless than 6 mV. There is no evidence of empty (non loaded) particlesbased on the manufacturing process.

TABLE 9 Quantitative Composition of ALN-VSP02 Component, gradeProportion (mg/mL) ALN-VSPDS01, cGMP 2.0* DLinDMA 7.3(1,2-Dilinoleyloxy- N,N-dimethyl-3-aminopropane), cGMP DPPC(R-1,2-Dipalmitoyl-sn-glycero-3- 1.1 phosphocholine), cGMP Cholesterol,Synthetic, cGMP 2.8 PEG2000-C-DMA 0.8 (3-N-[(ω-Methoxy poly(ethyleneglycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine), cGMP PhosphateBuffered Saline, cGMP q.s. *The 1:1 molar ratio of the two siRNAs in thedrug product is maintained throughout the size distribution of the drugproduct particles.

Solutions of lipid (in ethanol) and ALN VSPDS01 drug substance (inaqueous buffer) are mixed and diluted to form a colloidal dispersion ofsiRNA lipid particles with an average particle size of approximately80-90 nm. This dispersion is then filtered through 0.45/0.2 μm filters,concentrated, and diafiltered by Tangential Flow Filtration. After inprocess testing and concentration adjustment to 2.0 mg/mL, the productis sterile filtered, aseptically filled into glass vials, stoppered,capped and placed at 5±3° C. The ethanol and all aqueous buffercomponents are USP grade; all water used is USP Sterile Water ForInjection grade. Representative ALN-VSP02 process is shown in flowdiagram in FIG. 18.

9.4 Container/Closure System

The ALN VSP02 drug product is packaged in 10 mL glass vials with a fillvolume of 5 mL. The container closure system is comprised of a USP/EPType I borosilicate glass vial, a teflon faced butyl rubber stopper andan aluminum flip off cap. The drug product will be stored at 5±3° C.

9.5 Stability of Drug Product ALN-VSP02

The stability of the drug product was assayed at both storage conditions(2-8° C.) and at 25° C./60% RH. The drug product was stable at storagetemperature of 4° C. at 18 months (data not shown).

Example 10 In Vitro Efficacy of ALN-VSP02 in Human Cancer Cell Lines

The efficacy of ALN-VSP02 treatment in human cancer cell lines wasdetermined via measurement of KSP mRNA, VEGF mRNA, and cell viabilityafter treatment. IC50 (nM) values determined for KSP and VEGF in eachcell line.

TABLE 11 cell lines Cell line tested ATCC cat number HELA ATCC Cat N:CCL-2 KB ATCC Cat N: CCL-17 HEP3B ATCC Cat N: HB-8064 SKOV-3 ATCC Cat N:HTB-77 HCT-116 ATCC Cat N: CCL-247 HT-29 ATCC Cat N: HTB-38 PC-3 ATCCCat N: CRL-1435 A549 ATCC Cat N: CCL-185 MDA-MB-231 ATCC Cat N: HTB-26

Cells were plated in 96 well plates in complete media at day 1 to reacha density of 70% on day 2. On day 2 media was replaced with Opti-MEMreduced serum media (Invitrogen Cat N: 11058-021) and cells weretransfected with either ALN-VSP02 or control SNALP-Luc withconcentration range starting at 1.8 μM down to 10 pM. After 6 hours themedia was changed to complete media. Three replicate plates for eachcell line for each experiment was done.

Cells were harvested 24 hours after transfection. KSP levels weremeasured using bDNA; VEGF mRNA levels were measured using human TaqManassay.

Viability was measured using Cell Titer Blue reagent (Promega Cat N:G8080) at 48 and/or 72 h following manufacturer's recommendations.

As shown in Table 12, nM concentrations of VSP02 are effective inreducing expression of both KSP and VEGF in multiple human cell lines.Viability of treated cells was not

TABLE 12 Results Cell line IC50 (nM) KSP IC50 (nM) VEGF HeLa 8.79 672SKOV-3 142 1347 HCT116 31.6 27.5 Hep3B 1.3 14.5 HT-29 262 ND PC3 127 NDKB 50.6 ND A549 201 ND MB231 187 ND

Example 11 Anti-Tumor Efficacy of VSP SNALP Vs. Sorafenib in EstablishedHep3B Intrahepatic Tumors

The anti-tumor effects of multi-dosing VSP SNALP verses Sorafenib inscid/beige mice bearing established Hep3B intrahepatic tumors wasstudied. Sorafenib is a small molecule inhibitor of protein kinasesapproved for treatment of hepatic cellular carcinoma (HCC).

Tumors were established by intrahepatic seeding in scid/beige mice asdescribed herein. Treatment was initiated 11 days post-seeding. Micewere treated with Sorafenib and a control siRNA-SNALP, Sorafenib and VSPsiRNA-SNALP, or VSP siRNA-SNALP only. Control mice were treated withbuffers only (DMSO for Sorafenib and PBS for siRNA-SNALP). Sorafenib wasadministered intraparenterally from Mon to Fri for three weeks, at 15mg/kg according to body weight for a total of 15 injections. Sorafenibwas administered a minimum of 1 hour after SNALP injections. ThesiRNA-SNALPS were administered intravenously via the lateral tail veinaccording at 3 mg/kg based on the most recently recorded body weight (10ml/kg) for 3 weeks (total of 6 doses) on days 1, 4, 7, 10, 14, and 17.

Mice were euthanized based on an assessment of tumor burden includingprogressive weight loss and clinical signs including condition,abdominal distension/discoloration and mobility.

The percent survival data are shown in FIG. 21. Co-administration of VSPsiRNA-SNALP with Sorafenib increased survival proportion compared toadministration of Sorafenib or VSP siRNA-SNALP alone. VSP siRNA-SNALPincreased survival proportion compared to Sorafenib.

Example 12 In Vitro Efficacy of VSP Using Variants of AD-12115 andAD-3133

Two sets of duplexes targeted to Eg5/KSP and VEGF were designed andsynthesized. Each set included duplexes tiling 10 nucleotides in eachdirection of the target sites for either AD-12115 and AD-3133.

Sequences of the target, sense strand, and antisense strand for eachduplex are shown in the Table below.

Each duplex is assayed for inhibition of expression using the assaysdescribed herein. The duplexes are administered alone and/or incombination, e.g., an Eg5/KSP dsRNA in combination with a VEGF dsRNA. Insome embodiments, the dsRNA are administered in a SNALP formulation asdescribed herein.

TABLE 13 Sequences of dsRNA targeted to VEGF and Eg5/KSP (tiling) SEQSense Strand SEQ target target sequence ID Antisense strand ID Duplex IDgene 5′ to 3′ NO: 5′ to 3′ NO: AD-20447.1 VEGFA ACCAAGGCCAGCACAUAGG 14AccAAGGccAGcAcAuAGGTsT  54 CCuAUGUGCUGGCCUUGGUTsT  55 AD-20448.1 VEGFACCAAGGCCAGCACAUAGGA 15 ccAAGGccAGcAcAuAGGATsT  56 UCCuAUGUGCUGGCCUUGGTsT 57 AD-20449.1 VEGFA CCAAGGCCAGCACAUAGGA 16 ccAAGGccAGcAcAuAGGATsT  58CUCCuAUGUGCUGGCCUUGTsT  59 AD-20450.1 VEGFA AAGGCCAGCACAUAGGAGA 17AAGGccAGcAcAuAGGAGATsT  60 UCUCCuAUGUGCUGGCCUUTsT  61 AD-20451.1 VEGFAAGGCCAGCACAUAGGAGAG 18 AGGccAGcAcAuAGGAGAGTsT  62 CUCUCCuAUGUGCUGGCCUTsT 63 AD-20452.1 VEGFA GGCCAGCACAUAGGAGAGA 19 GGccAGcAcAuAGGAGAGATsT  64UCUCUCCuAUGUGCUGGCCTsT  65 AD-20453.1 VEGFA GCCAGCACAUAGGAGAGAU 20GccAGcAcAuAGGAGAGAuTsT  66 AUCUCUCCuAUGUGCUGGCTsT  67 AD-20454.1 VEGFACCAGCACAUAGGAGAGAUG 21 ccAGcAcAuAGGAGAGAuGTsT  68 cAUCUCUCCuAUGUGCUGGTsT 69 AD-20455.1 VEGFA CAGCACAUAGGAGAGAUGA 22 cAGcAcAuAGGAGAGAuGATsT  70UcAUCUCUCCuAUGUGCUGTsT  71 AD-20456.1 VEGFA AGCACAUAGGAGAGAUGAG 23AGcAcAuAGGAGAGAuGAGTsT  72 CUcAUCUCUCCuAUGUGCUTsT  73 AD-20457.1 VEGFACACAUAGGAGAGAUGAGCU 24 cAcAuAGGAGAGAuGAGcuTsT  74 AGCUcAUCUCUCCuAUGUGTsT 75 AD-20458.1 VEGFA ACAUAGGAGAGAUGAGCUU 25 AcAuAGGAGAGAuGAGcuuTsT  76AAGCUcAUCUCUCCuAUGUTsT  77 AD-20459.1 VEGFA CAUAGGAGAGAUGAGCUUC 26cAuAGGAGAGAuGAGcuucTsT  78 GAAGCUcAUCUCUCCuAUGTsT  79 AD-20460.1 VEGFAAUAGGAGAGAUGAGCUUCC 27 AuAGGAGAGAuGAGcuuccTsT  80 GGAAGCUcAUCUCUCCuAUTsT 81 AD-20461.1 VEGFA UAGGAGAGAUGAGCUUCCU 28 uAGGAGAGAuGAGcuuccuTsT  82AGGAAGCUcAUCUCUCCuATsT  83 AD-20462.1 VEGFA AGGAGAGAUGAGCUUCCUA 29AGGAGAGAuGAGcuuccuATsT  84 uAGGAAGCUcAUCUCUCCUTsT  85 AD-20463.1 VEGFAGGAGAGAUGAGCUUCCUAC 30 GGAGAGAuGAGcuuccuAcTsT  86 GuAGGAAGCUcAUCUCUCCTsT 87 AD-20464.1 VEGFA GAGAGAUGAGCUUCCUACA 31 GAGAGAuGAGcuuccuAcATsT  88UGuAGGAAGCUcAUCUCUCTsT  89 AD-20465.1 VEGFA AGAGAUGAGCUUCCUACAG 32AGAGAuGAGcuuccuAcAGTsT  90 CUGuAGGAAGCUcAUCUCUTsT  91 AD-20466.1 VEGFAGAGAUGAGCUUCCUACAGC 33 GAGAuGAGcuuccuAcAGcTsT  92 GCUGuAGGAAGCUcAUCUCTsT 93 AD-20467.1 KSP AUGUUCCUUAUCGAGAAUC 34 AuGuuccuuAucGAGAAucTsT  94GAUUCUCGAuAAGGAAcAUTsT  95 AD-20468.1 KSP UGUUCCUUAUCGAGAAUCU 35uGuuccuuAucGAGAAucuTsT  96 AGAUUCUCGAuAAGGAAcATsT  97 AD-20469.1 KSPGUUCCUUAUCGAGAAUCUA 36 GuuccuuAucGAGAAucuATsT  98 uAGAUUCUCGAuAAGGAACTsT 99 AD-20470.1 KSP UUCCUUAUCGAGAAUCUAA 37 uuccuuAucGAGAAucuAATsT 100UuAGAUUCUCGAuAAGGAATsT 101 AD-20471.1 KSP UCCUUAUCGAGAAUCUAAA 38uccuuAucGAGAAucuAAATsT 102 UUuAGAUUCUCGAuAAGGATsT 103 AD-20472.1 KSPCCUUAUCGAGAAUCUAAAC 39 ccuuAucGAGAAucuAAAcTsT 104 GUUuAGAUUCUCGAuAAGGTsT105 AD-20473.1 KSP CUUAUCGAGAAUCUAAACU 40 cuuAucGAGAAucuAAAcuTsT 106AGUUuAGAUUCUCGAuAAGTsT 107 AD-20474.1 KSP UUAUCGAGAAUCUAAACUA 41uuAucGAGAAucuAAAcuATsT 108 uAGUUuAGAUUCUCGAuAATsT 109 AD-20475.1 KSPUAUCGAGAAUCUAAACUAA 42 uAucGAGAAucuAAAcuAATsT 110 UuAGUUuAGAUUCUCGAuATsT111 AD-20476.1 KSP AUCGAGAAUCUAAACUAAC 43 AucGAGAAucuAAAcuAAcTsT 112GUuAGUUuAGAUUCUCGAUTsT 113 AD-20477.1 KSP CGAGAAUCUAAACUAACUA 44cGAGAAucuAAAcuAAcuATsT 114 uAGUuAGUUuAGAUUCUCGTsT 115 AD-20478.1 KSPGAGAAUCUAAACUAACUAG 45 GAGAAucuAAAcuAAcuAGTsT 116 CuAGUuAGUUuAGAUUCUCTsT117 AD-20479.1 KSP AGAAUCUAAACUAACUAGA 46 AGAAucuAAAcuAAcuAGATsT 118UCuAGUuAGUUuAGAUUCUTsT 119 AD-20480.1 KSP GAAUCUAAACUAACUAGAA 47GAAucuAAAcuAAcuAGAATsT 120 UUCuAGUuAGUUuAGAUUCTsT 121 AD-20481.1 KSPAAUCUAAACUAACUAGAAU 48 AAucuAAAcuAAcuAGAAuTsT 122 AUUCuAGUuAGUUuAGAUUTsT123 AD-20482.1 KSP AUCUAAACUAACUAGAAUC 49 AucuAAAcuAAcuAGAAucTsT 124GAUUCuAGUuAGUUuAGAUTsT 125 AD-20483.1 KSP UCUAAACUAACUAGAAUCC 50ucuAAAcuAAcuAGAAuccTsT 126 GGAUUCuAGUuAGUUuAGATsT 127 AD-20484.1 KSPCUAAACUAACUAGAAUCCU 51 cuAAAcuAAcuAGAAuccuTsT 128 AGGAUUCuAGUuAGUUuAGTsT129 AD-20485.1 KSP UAAACUAACUAGAAUCCUC 52 uAAAcuAAcuAGAAuccucTsT 130GAGGAUUCuAGUuAGUUuATsT 131 AD-20486.1 KSP AAACUAACUAGAAUCCUCC 53AAAcuAAcuAGAAuccuccTsT 132 GGAGGAUUCuAGUuAGUUUTsT 133

Example 13 VEGF Targeted dsRNA with a Single Blunt End

A set duplexes targeted to VEGF were designed and synthesized. The setincluded duplexes tiling 10 nucleotides in each direction of the targetsites for AD-3133. Each duplex includes a 2 base overhang at the endcorresponding to the 3′ end of the antisense strand and no overhang,e.g., a blunt end, at the end corresponding to the 5′ end of theantisense strand.

The sequences of each strand of these duplexes are shown in thefollowing table.

Each duplex is assayed for inhibition of expression using the assaysdescribed herein. The VEGF duplexes are administered alone and/or incombination with an Eg5/KSP dsRNA (e.g., AD-12115). In some embodiments,the dsRNA are administered in a SNALP formulation as described herein.

TABLE 14 Target sequences of blunt ended dsRNA targeted to VEGF SEQVEGF target position duplex ID sequence on VEGF ID NO: 5′ to 3′ geneAD-20447.1 134 ACCAAGGCCAGCACAUAGG 1365 AD-20448.1 135CCAAGGCCAGCACAUAGGA 1366 AD-20449.1 136 CAAGGCCAGCACAUAGGAG 1367AD-20450.1 137 AAGGCCAGCACAUAGGAGA 1368 AD-20451.1 138AGGCCAGCACAUAGGAGAG 1369 AD-20452.1 139 GGCCAGCACAUAGGAGAGA 1370AD-20453.1 140 GCCAGCACAUAGGAGAGAU 1371 AD-20454.1 141CCAGCACAUAGGAGAGAUG 1372 AD-20455.1 142 CAGCACAUAGGAGAGAUGA 1373AD-20456.1 143 AGCACAUAGGAGAGAUGAG 1374 AD-20457.1 144CACAUAGGAGAGAUGAGCU 1376 AD-20458.1 145 ACAUAGGAGAGAUGAGCUU 1377AD-20459.1 146 CAUAGGAGAGAUGAGCUUC 1378 AD-20460.1 147AUAGGAGAGAUGAGCUUCC 1379 AD-20461.1 148 UAGGAGAGAUGAGCUUCCU 1380AD-20462.1 149 AGGAGAGAUGAGCUUCCUA 1381 AD-20463.1 150GGAGAGAUGAGCUUCCUAC 1382 AD-20464.1 151 GAGAGAUGAGCUUCCUACA 1383AD-20465.1 152 AGAGAUGAGCUUCCUACAG 1384 AD-20466.1 153GAGAUGAGCUUCCUACAGC 1385

TABLE 15 Strand sequences of blunt ended dsRNA targeted to VEGF SEQ SEQduplex Sense strand ID Antisense strand ID ID (5′ to 3′) NO: (5′ to 3′)NO: AD-20447.1 ACCAAGGCCAGCACAUAGGAG 154 CUCCUAUGUGCUGGCCUUGGUGA 155AD-20448.1 CCAAGGCCAGCACAUAGGAGA 156 UCUCCUAUGUGCUGGCCUUGGUG 157AD-20449.1 CAAGGCCAGCACAUAGGAGAG 158 CUCUCCUAUGUGCUGGCCUUGGU 159AD-20450.1 AAGGCCAGCACAUAGGAGAGA 160 UCUCUCCUAUGUGCUGGCCUUGG 161AD-20451.1 AGGCCAGCACAUAGGAGAGAU 162 AUCUCUCCUAUGUGCUGGCCUUG 163AD-20452.1 GGCCAGCACAUAGGAGAGAUG 164 CAUCUCUCCUAUGUGCUGGCCUU 165AD-20453.1 GCCAGCACAUAGGAGAGAUGA 166 UCAUCUCUCCUAUGUGCUGGCCU 167AD-20454.1 CCAGCACAUAGGAGAGAUGAG 168 CUCAUCUCUCCUAUGUGCUGGCC 169AD-20455.1 CAGCACAUAGGAGAGAUGAGC 170 GCUCAUCUCUCCUAUGUGCUGGC 171AD-20456.1 AGCACAUAGGAGAGAUGAGCU 172 AGCUCAUCUCUCCUAUGUGCUGG 173AD-20457.1 CACAUAGGAGAGAUGAGCUUC 174 GAAGCUCAUCUCUCCUAUGUGCU 175AD-20458.1 ACAUAGGAGAGAUGAGCUUCC 176 GGAAGCUCAUCUCUCCUAUGUGC 177AD-20459.1 CAUAGGAGAGAUGAGCUUCCU 178 AGGAAGCUCAUCUCUCCUAUGUG 179AD-20460.1 AUAGGAGAGAUGAGCUUCCUA 180 UAGGAAGCUCAUCUCUCCUAUGU 181AD-20461.1 UAGGAGAGAUGAGCUUCCUAC 182 GUAGGAAGCUCAUCUCUCCUAUG 183AD-20462.1 AGGAGAGAUGAGCUUCCUACA 184 UGUAGGAAGCUCAUCUCUCCUAU 185AD-20463.1 GGAGAGAUGAGCUUCCUACAG 186 CUGUAGGAAGCUCAUCUCUCCUA 187AD-20464.1 GAGAGAUGAGCUUCCUACAGC 188 GCUGUAGGAAGCUCAUCUCUCCU 189AD-20465.1 AGAGAUGAGCUUCCUACAGCA 190 UGCUGUAGGAAGCUCAUCUCUCC 191AD-20466.1 GAGAUGAGCUUCCUACAGCAC 192 GUGCUGUAGGAAGCUCAUCUCUC 193

Example 14 Inhibition of Eg5/KSP and VEGF Expression in Humans

A human subject is treated with a pharmaceutical composition, e.g.,ALN-VSP02, having both a SNALP formulated dsRNA targeted to a Eg5/KSPgene and a SNALP formulated dsRNA targeted to a VEGF gene to inhibitexpression of the Eg5/KSP and VEGF genes.

A subject in need of treatment is selected or identified. The subjectcan be in need of cancer treatment, e.g., liver cancer.

At time zero, a suitable first dose of the composition is subcutaneouslyadministered to the subject. The composition is formulated as describedherein. After a period of time, the subject's condition is evaluated,e.g., by measurement of tumor growth, measuring serum AFP levels, andthe like. This measurement can be accompanied by a measurement ofEg5/KSP and/or VEGF expression in said subject, and/or the products ofthe successful siRNA-targeting of Eg5/KSP and/or VEGF mRNA. Otherrelevant criteria can also be measured. The number and strength of dosesare adjusted according to the subject's needs.

After treatment, the subject's condition is compared to the conditionexisting prior to the treatment, or relative to the condition of asimilarly afflicted but untreated subject.

Example 15 Clinical Trial of ALN-VSP02 in Humans

A clinical study is performed to assess the safety and tolerability ofALN-VSP02 in patients with advanced solid tumors with liver involvement,to characterize the PK (pharmacokinetics) of ALN-VSP02, and to assesspreliminary evidence of antitumor/antiangiogenic activity of ALN-VSP02.The study is an open label, multi-center, with dose-escalation,utilizing up to ˜55 patients with primary or secondary liver cancer.

ALN-VSP02 is administered by 15 minute IV infusion every 2 weeks at doselevels: 0.1, 0.2, 0.3, 0.4, 0.7, 1.0, 1.25, 1.5, 1.7, 2.0, 3.0, and 6.0mg/kg.

Tumor measurements are performed after every 4 doses. Patients continuetherapy until disease progression (as defined by RECIST criteria) orunacceptable toxicity is reached.

Example 16 Comparison of PK Data for ALN-12115 from Phase 1 Study:ALN-VSP02 in Humans

A clinical study was begun using the parameters described in Example 15.Three core biopsies were taken per time point from single tumor:pre-treatment and 2 and 7 days post-dose 1. Each biopsy was processed by2 methods: 1: formalin-fixed, paraffin-embedded and 2, snap-frozen inliquid nitrogen. The PK of ALN-12115 was quantified in the tumors usingqRT-PCR.

Allometric scaling and the predicted AUC (are under curve) of ALN-12115(the KSP duplex in ALN-VSP02) are described as follows. PredictedC_(max), and AUC_(n-∞) will have actual response ranging from 1.5 to 3.4times of their predicted values; that is, the error in predicted dosewill not be greater than ±)3- to 4-fold (Mordenti et al.).

Prediction in an 85 kg Unit Allometric Equation Human CL mL/min LogCL =1.04 · LogBW + 0.01  103 (1.21 mL/min/kg) Vd mL LogVd = 0.98 · LogBW +1.98 7394 (86.99 mL/kg)

-   -   Predicted AUC=(D×F)/CL in human (F=1 for IV dosing)

Phase 1 Infusion Predicted Predicted HED Exposure Dose Human Vd Human CL(AUC) mg/kg mg mL mL/min ng * min/mL 0.05 4.25 7394 103 41202 0.1 8.57394 103 82404 0.2 17 7394 103 164808 0.4 34 7394 103 329616 0.7 59.57394 103 576828

A comparision of PK data for ALN-12115 obtained from the phase 1 studyis shown below. For Cohort 1, the predicted AUC was 41202 ng·min/mLwhile actual mean ranged 30784 to 37280 ng·min/mL. For cohort 2, thepredicted AUC was 82404 ng·min/mL while actual mean ranged from 115469to 130736 ng·min/mL. The AUC is within the predicted value of notgreater than (±) 3 to 4 fold.

Cohort 1: 0.1 mg/kg C1W1 C2W2 Parameter 001-002 002-001 002-003 Mean001-002 002-001 Mean Cmax (ng/mL) 519 1167 583 756 673 1181 927AUC_(0-last) 11407 53178 27766 30784 21198 53363 37280 (ng * min/mL)AUC_(partial (0-135 min)) 11407 42312 22596 25558 21198 45331 33265(ng * min/mL) t½α (min) 18.2 18.6 15.5 17.4 15.6 18.2 16.9

Cohort 2: 0.2 mg/kg C1W1 C2W2 Parameter 002-004 002-006 003-005 Mean002-004 002-006 003-005 Mean Cmax (ng/mL) 2266 2803 1723 2264 2684 21111921 2239 AUC_(0-last) 173850 134162 84197 130736 181038 110683 54686115469 (ng * min/mL) AUC_(partial (0-135 min)) 113685 124975 81186106615 127422 104407 52997 94942 (ng * min/mL) t½α (min) 25.2 19.8 14.819.9 20.5 17.7 15.0 17.7

ABBREVIATIONS

CL—clearance—volume of plasma from which the drug is completely removedper unit time. The amount eliminated is proportional to theconcentration of the drug in the blood.

Vd—Volume of distribution—amount of drug in the body divided by theconcentration in the blood.

AUC—total area under the curve—is very useful for calculating therelative efficiency of different drug products

C_(max)—highest concentration

t½—half-life—time required for a given drug concentration to decrease by50%. T½ is determined by the clearance and the volume of distribution.

C1W1—Cycle 1, week 1.

C2W2—Cycle 2, week 2.

Example 17 Treatment of Patients with Advanced Cancer with LiverInvolvement Using ALN-VSP02

A clinical study was performed to assess the safety and tolerability ofALN-VSP02 in patients with advanced solid tumors with liver involvement,to characterize the PK (pharmacokinetics) of ALN-VSP02, and to assesspreliminary evidence of antitumor/antiangiogenic activity of ALN-VSP02at various dosage levels. The study focused on patients with primary orsecondary liver cancer.

Patients having advanced cancer with liver involvement were given a 15minute IV infusion of ALN-VSP02 every 2 weeks at the following dosagelevels: 0.1, 0.2, 0.4, and 0.7 mg/kg. Treatments were given in cycles of2 doses (1 month), with tumor measurements taken after every 2 cycles.Following administration of each dose, plasma samples were taken atdefined time intervals and assayed for levels of ALN-VSP02 concentrationto obtain pharmacokinetic information and to observe any evidence ofdrug accumulation.

C_(max), t_(max), and AUC of KSP siRNA in the patient's plasma for thefirst dose and the third dose are shown in Tables 16a and 16brespectively. C_(max), t_(max), and AUC for VEGF siRNA in the patient'splasma for the first dose and the third dose are shown in Tables 17a and17b respectively. These tables show pharmacokinetic data from the first3 patients enrolled onto each of the dose levels, i.e. N=3 for the firstand third dose of ALN-VSP02. ALN-VSP02 treatment resulted indose-proportional C_(max) and AUC of ALN-VSP02 in human plasma with noevidence of drug accumulation between dose 1 and dose 3.

TABLE 16a KSP siRNA mean plasma concentration parameter estimates bydose level ALN-VSP Dose 1 (mg/kg) 0.1 0.2 0.4 0.7 C_(max) 0.76 ± 0.36 2.3 ± 0.54 3.2 ± 1.2 9.8 ± 4.1 (μg/mL) t_(max) (min) 18.3 ± 5.8  16.7 ±2.9 17 ± 3  20 ± 5  AUC_(0-last) 30.9 ± 21.1 130.7 ± 44.9 201.3 ± 38.6 501.2 ± 203.9 (μg · min/mL)

TABLE 16b KSP siRNA mean plasma concentration parameter estimates bydose level and evidence of drug accumulation ALN-VSP Dose 3 (mg/kg) 0.10.2 0.4 0.7 Cmax (μg/mL) 0.93  2.2 ± 0.40 4.8 9.3 tmax (min) 15 16.7 ±2.9 18 15 AUC0-last 37.3 130.7 ± 44.9 252.3 579.3 (μg · min/mL)

TABLE 17a VEGF siRNA mean plasma concentration parameter estimates bydose level. ALN-VSP Dose 1 (mg/kg) 0.1 0.2 0.4 0.7 Cmax 0.86 ± 0.43  2.5± 0.56  3.7 ± 1.2 9.7 ± 2.7 (μg/mL) tmax (min) 26.7 ± 5.8  21.7 ± 2.9 15± 0 18 ± 6  AUC0-last 36.9 ± 20.2 140.3 ± 56.1 207.7 ± 36.3 610.9 ±223.3 (ug · min/ mL)

TABLE 17b VEGF siRNA mean plasma concentration parameter estimates bydose level and evidence of drug accumulation ALN-VSP Dose 3 (mg/kg) 0.10.2 0.4 0.7 Cmax (μg/mL) 0.98  2.2 ± 0.36 5 8.8 tmax (min) 15 18.3 ± 2.918 20 AUC0-last 40.7 114.7 ± 62.9 330.9 622.4 (μg · min/mL)

DCE-MRI evaluation of one or more evaluable liver tumors was performedin patients at each dosage level. Measurements were obtained once beforetreatment (baseline), once on Day 3-5 post-dose, and once again on Day8-10 post-dose. The average change in K_(trans) from baseline wasderived form the peak change in K_(trans) for each evaluable tumorfollowing the 2 post-treatment scans. The results are suggestive of ananti-VEGF effect in the majority of treated patients. Some patients alsohad an associated reduction in plasma VEGF levels.

In the majority of patients, only mild drug-related adverse effects wereobserved at 0.1 to 0.7 mg/kg dosages, indicating that ALN-VSP02 isgenerally well tolerated at the doses provided.

The ALN-VSP02 composition is effective for treatment of patientsdiagnosed with cancer with liver involvement and is well tolerated inpatients at least up to dosages of 0.7 mg/kg. ALN-VSP-2 human plasmapharmacokinetics showed dose-proportional C_(max) and AUC with noevidence of drug accumulation.

Example 18 Pharmacokinetic Profile of ALN-VSP02 After 15-Minute IVInfusion in Patients with Advanced Solid Tumor

A clinical study was performed to characterize the PK (pharmacokinetics)of ALN-VSP02 in patients with advanced solid tumors with liverinvolvement.

41 patients having advanced cancer with liver involvement were given a15 minute IV infusion of ALN-VSP02 at the following dosage levels: 0.1,0.2, 0.4, 0.7, 1.0, 1.25 and 1.50 mg/kg. The median age of the patientswas 57 years (range 34-78, 17 males and 24 females) and all patients hadreceived multiple prior cancer therapies (average of 4.3 prior regimensfor meatstatic disease, range 1-13). A single dosage level defined acohort; n=3 patients per cohort. Treatments were provided once every 2weeks, with 1 cycle comprised of 2 doses. A pharmacokinetic evaluationwas performed after the first dose (C1W1D1) and after the third dose(C2W1D1). A single patient remains on the study treatment after 1 yearof dosing.

Plasma samples were taken at defined time intervals and assayed forlevels of ALN-VSP02 concentration to obtain pharmacokinetic informationand to observe any evidence of drug accumulation. Plasma samples wereobtained at Pre-dose, end of infusion, and at 5, 10 and 30 minutes and2, 4, 6, 8 and 24 hours post-end of infusion. Both total and free siRNA(siRNA encapsulated and un-encapsulated) were measured.

Urine samples were obtained 0-8 and 8-24 hours post-end of infusion andanalyzed by hybridization using the following lower limit ofquantification (LLOQ): LLOQ at 3 ng/mL for AD-3133 (siRNA targetingVEGF) and LLOQ at 1 ng/mL for AD-12115 (siRNA targeting KSP/Eg5). Allpre-dose siRNA concentrations were <LLOQ.

Plasma concentration-time profiles were generated for each siRNA. Theconcentration-time profiles obtained were used to calculate thepharmacokinetic (PK) parameter estimates using WinNonlin® V5.2.1software for PK/PD modeling (Pharsight®, St. Louis, Mo.).

Results

The plasma concentration time profiles for all 7 dosage levels/cohortshad similar properties: all were multiphasic, with a rapid distributionphase and similar profiles at C1 (first dose) and C2 (third dose) (datanot shown). For a1

1 cohorts, the PK parameters showed that the C_(max) was approximatelysimilar at C1 and C2 and t_(1/2α) was short and approximately similar atC1 and C2. In some patients at some dosages, the AUC was approximatelysimilar at C1 and C2. For Cohorts 1, 4, and 5 the elimination phase wasnot fully captured due to assay sensitivity

The mean plasma PK parameters are provided in Tables 18 and 19.

TABLE 18 Mean Plasma PK Parameters for AD-12115 KSP siRNA 0.1 mg/kg 0.2mg/kg 0.4 mg/kg 0.7 mg/kg 1.0 mg/kg 1.25 mg/kg 1.5 mg/kg (AD-12115) (n =3) (n = 3) (n = 5) (n = 7) (n = 4) (n = 5) (n = 2) Cycle 1 (C1W1)T_(max) 0.31 0.28 0.38 0.31 0.36 0.46 0.29 (hr) (0.10) (0.05) (0.13)(0.08) (0.09) (0.19) (NA) C_(max) 756.28 2263.95 3918.17 7990.7315314.98 21937.49 18042.67 (ng/mL) (356.85) (539.67) (1115.47) (3389.68)(5347.86) (7642.00) (NA) AUC_(0-last) 513.90 2216.95 4206.32 8719.6315393.10 33929.01 24850.50 (ng * hr/mL) (347.65) (777.70) (1631.70)(3436.35) (6002.12) (18334.75) (NA) Cycle 2 (C2W1) T_(max) 0.15 0.280.33 0.30 0.31 0.29 0.42 (hr) (NA) (0.05) (0.07) (0.09) (0.08) (NA) (NA)C_(max) 657.99 2239.05 3968.35 8399.62 12440.80 17897.05 23197.47(ng/mL) (NA) (397.66) (886.88) (2565.51) (9615.15) (NA) (NA)AUC_(0-last) 649.22 1833.88 4653.68 11148.85 9984.02 28833.57 27661.82(ng * hr/mL) (NA) (869.14) (2349.91) (3453.67) (2517.30) (NA) (NA) (NA):Not Available Standard deviation in parentheses

TABLE 19 Mean Plasma PK Parameters for AD-3133 VEGF siRNA 0.1 mg/kg 0.2mg/kg 0.4 mg/kg 0.7 mg/kg 1.0 mg/kg 1.25 mg/kg 1.5 mg/kg (AD-3133) (n =3) (n = 3) (n = 5) (n = 7) (n = 4) (n = 5) (n = 2) Cycle 1 (C1W1)T_(max) 0.31 0.36 0.38 0.31 0.37 0.34 0.29 (hr) (0.10) (0.05) (0.13)(0.08) (0.10) (0.10) (NA) C_(max) 862.42 2509.81 4305.06 8805.5815843.73 21578.94 18118.21 (ng/mL) (427.78) (559.82) (1096.47) (2747.25)(7014.77) (6798.07) (NA) AUC_(0-last) 1149.10 2385.18 4536.91 10439.4616404.33 33442.95 25243.90 (ng * hr/mL) (1101.94) (976.34) (1636.03)(4544.30) (7280.14) (18558.98) (NA) Cycle 2 (C2W1) T_(max) 0.25 0.320.36 0.32 0.29 0.29 0.42 (hr) (NA) (0.06) (0.09) (0.09) (0.04) (NA) (NA)C_(max) 976.45 2188.67 4408.93 8595.47 12681.94 18230.74 22531.47(ng/mL) (NA) (360.97) (616.44) (2070.75) (8811.54) (NA) (NA)AUC_(0-last) 678.70 1908.36 5388.18 12002.74 9563.98 31072.98 28498.06(ng * hr/mL) (NA) (997.93) (2752.38) (3475.86) (2173.22) (NA) (NA) (NA):Not Available Standard deviation in parentheses

In general, concentration of urine siRNA was detectable at 0-8 hours forboth siRNA in both cohorts, and levels decreased at 8-24 hours.

The AD-12115 and AD-3133 plasma concentration-time profiles weremultiphasic and peaked at approximately end of infusion. The C_(max) andAUC_(0-last) approximately increased proportionally with increases indose after a single or multiple administrations at 0.1, 0.2, 0.4, 0.7,1.0, 1.25 and 1.5 mg/kg of ALN-VSP02.

Maximal exposure to ALN-VSP02 was produced at 1.5 mg/kg. A similarexposure of AD-12115 and AD-3133 was observed at C1W1 (1^(st) dose)compared to C2W2 (3^(rd) dose).

At a dosage of 1.0 mg/kg, the mean C_(max) after the first dose (C1W1)for AD-12115 was 15314.98±5347.86 ng/mL and for AD-3133 was15843.73±7014.77 ng/mL. The mean C_(max) after the second dose (C2W1)for AD-12115 was 12440.80±9615.15 ng/mL and for AD-3133 was12681.94±8811.54 ng/mL. The mean AUC_(0-last) after the first dose(C1W1) for AD-3133 was 16404.33±7280.14 ng*hr/mL and for AD-12115 was15393.10±6002.12 ng*hr/mL. The mean AUC_(0-last) after second dose(C2W1) for AD-3133 was 9563.98±2173.22 ng*hr/mL and for AD-12115 was9984.02±2517.30 ng*hr/mL.

The mean distribution half life (T_(1/2α)) for ALN-12115 and ALN-3133ranged from 21.41 to 74.93 minutes and from 12.87 to 34.83 minutes forC1W1, respectively.

The mean distribution half life (T_(1/2α)) for ALN-12115 and ALN-3133ranged from 13.70 to 43.46 minutes and from 17.14 to 44.28 minutes forC2W2, respectively.

Table 20 shows a summary of the patient's tumor response.

Number of Patients Number of Average with Stable Number of Patients DosePatients Number Disease or Who Proceeded to Level Evaluable for of DosesBetter for 2 Extension Study (>8 (mg/kg) Response (Range) ≧Months Doses)To Date 0.1 3   3 (2-4) 0 0 0.2 3   4 (4-4) 0 0 0.4  7* 4.6 (2-11) 1 10.7 5 9.6 (3-23)   3*** 2 1.0  11** 4.8 (2-8) 7 1 1.25 7 2.4 (1-6) 2 01.5 1 4 0 0 *Includes 1 patient whose first dose was 0.7 mg/kg**Includes 2 patient whose first dose was 1.25 mg/kg ***Includes 1patial response with ~70% tumor reduction

The disease was stable or better in 50% (12/24) of the patients who wereadministered a dose of at least 0.7 mg/kg.

DCE-MRI evaluation of one or more evaluable liver tumors was performedin patients at each dosage level. Measurements were obtained once beforetreatment (baseline), once on Day 3-5 post-dose, and once again on Day8-10 post-dose. The average change in K_(trans) from baseline wasderived form the peak change in K_(trans) for each evaluable tumorfollowing the 2 post-treatment scans. 46% (13/28) of the patients showedan average decrease in K_(trans) in liver tumors of ≧40%.

29 tumor biopsies were obtained from 15 patients. The results of thosebiopsies are provided in Table 21 below. These biopsies were tested forVEGF by 5′ RACE. 3 of 15 patients were positive for VEGF 5′ RACE.

TABLE 21 Tumor Type Post-Dose Drug Levels** Patient No./ (Biopsy Site,Biopsy (%) (ng/g tissue) Dose Day of Post-Dose Viable Fibrosis/ VEGF KSPmg/kg Biopsy) Tumor Liver Necrosis siRNA siRNA A01/ Colorectal (liver,d7) 17 80  3 25.7 12.5 0.4 A02/ Ovarian (liver, d2) 0 95  5 28.9 17.20.4 A03/ Colorectal (liver, d2) 20 5 75 142 73.3 0.7 A04/ Colorectal(adrenal, d2) 10 0 78* 9.8 3.8 1.0 A05/ Sarcoma (muscle, d2) 96 0  40.45 0.40 1.0 A06/ Colorectal (liver, d3) 56 0 35^(†) 5.4 2.2 1.0 A07/RCC (liver, d2) 70 15 15 6.0 6.8 1.0 A08/ Uveal mel (liver, d2) 100 0  07.0 5.8 1.0 A09/ Colorectal (liver, d6) 14 0 71^(‡) 0.32 <LLOQ 1.25 A10/Ovarian (abdomen, d4) 30 0 70 4.9 3.6 1.25 A11/ Ovarian (L. node, d5) 400 60 20.4 20.3 1.25 A12/ Small bowel (liver, d6) 42 0 58 4.9 3.6 1.5

Those skilled in the art are familiar with methods and compositions inaddition to those specifically set out in the present disclosure whichwill allow them to practice this invention to the full scope of theclaims hereinafter appended.

What is claimed is:
 1. A method of treating endometrial cancer in asubject in need thereof, comprising administering to the subject atleast about 0.7 mg/kg (based on the total weight of siRNA in mg per kgof body weight) of a composition comprising: (i) DLinDMA; (ii) DPPC;(iii) PEG2000-C-DMA; (iv) cholesterol, (v) an siRNA targeting Eg5/KSP;and (vi) an siRNA targeting VEGF.
 2. The method of claim 1, wherein thesubject has previously failed to respond to cancer treatment.
 3. Themethod of claim 1, wherein the siRNA targeting Eg5/KSP is AD12115. 4.The method of claim 1, wherein the siRNA targeting VEGF is AD3133. 5.The method of claim 1, wherein about 1.0 mg/kg of the composition isadministered.
 6. The method of claim 1, wherein at least about 1.0 mg/kgof the composition is administered.
 7. The method of claim 1, whereinthe subject has advanced cancer with liver involvement.
 8. The method ofclaim 1, wherein the composition is administered by intravenous (IV)infusion once every two weeks.
 9. The method of claim 8, wherein theduration of each IV infusion ranges from about 15 minutes to about 3hours.
 10. The method of claim 1, wherein the composition isadministered to the subject once every 2 weeks for at least four weeks.11. The method of claim 1, wherein the molar ratio ofDLinDMA:DPPC:cholesterol:PEG2000-C-DMA is about 57.1:7.1:34.4:1.4. 12.The method of claim 1, wherein the molar ratio of siRNA targetingEg5/KSP to siRNA targeting VEGF is about 1 to
 1. 13. The method of claim1, further comprising administering a corticosteroid to the subject. 14.The method of claim 1, wherein the composition is ALN-VSP02 and providesa mean KSP siRNA AUC_(0-last) from about 10 to about 1100 μg*min/mL, amean KSP siRNA C_(max) from about 0.4 to about 10.0 μg/mL, a mean VEGFsiRNA AUC_(0-last) from about 10 to about 1200 μg*min/mL and a mean VEGFsiRNA C_(max) from about 0.4 to about 18 μg/mL.
 15. The method of claim1, wherein following administration of a first dose of 1 mg/kg of thecomposition, the AUC_(0-last) of KSP siRNA is within about 80% to about125% of 15393.10±6002.12 ng*hr/mL, and/or following administration of asecond dose of 1 mg/kg of the composition, the AUC_(0-last) of KSP siRNAis within about 80% to about 125% of 9984.02±2517.30 ng*hr/mL.
 16. Themethod of claim 1, wherein following administration of a first dose of 1mg/kg of the composition, the C_(max) of KSP siRNA is within about 80%to about 125% of 15314.98±5347.86 ng/mL, and/or following administrationof a second dose of 1 mg/kg of the composition, the C_(max) of KSP siRNAis within about 80% to about 125% of 12440.80±9615.15 ng/mL.
 17. Themethod of claim 1, wherein following administration of a first dose of 1mg/kg of the composition, the AUC_(0-last) of VEGF siRNA is within about80% to about 125% of 16404.33±7280.14 ng*hr/mL, and/or followingadministration of a second dose of 1 mg/kg of the composition, theAUC_(0-last) of VEGF siRNA is within about 80% to about 125% of9563.98±2173.22 ng*hr/mL.
 18. The method of claim 1, wherein followingadministration of a first dose of 1 mg/kg of the composition, theC_(max) of VEGF siRNA is within about 80% to about 125% of15843.73±7014.77 ng/mL, and/or following administration of a second doseof 1 mg/kg of the composition, the C_(max) of VEGF siRNA is within about80% to about 125% of 12681.94±8811.54 ng/mL.
 19. A method of treating asubject in need of treatment, comprising administering to the subject a1.0 mg/kg dosage of a composition comprising (i) DLinDMA; (ii) DPPC;(iii) PEG2000-C-DMA; (iv) cholesterol, (v) an siRNA targeting Eg5/KSP;and (vi) an siRNA targeting VEGF; via intravenous (IV) infusion onceevery 2 weeks, wherein the composition has a dose-proportional maximumconcentration (C_(max)) and area under curve (AUC) as measurable in thesubject's plasma; the AUC_(0-last) of KSP siRNA is within about 80% toabout 125% of 15393.10±6002.12 or 9984.02±2517.30 ng*hr/mL; the C_(max)of KSP siRNA is within about 80% to about 125% of 15314.98±5347.86 or12440.80±9615.15 ng/mL; the AUC_(0-last) of VEGF siRNA is within about80% to about 125% of 16404.33±7280.14 or 9563.98±2173.22 ng*hr/mL; andthe C_(max) of VEGF siRNA is within about 80% to about 125% of15843.73±7014.77 or 12681.94±8811.54 ng/mL.
 20. A method of treating asubject in need of treatment, comprising administering to the subject a1.25 mg/kg dosage of a composition comprising (vii) DLinDMA; (viii)DPPC; (ix) PEG2000-C-DMA; (x) cholesterol, (xi) an siRNA targetingEg5/KSP; and (xii) an siRNA targeting VEGF; via intravenous (IV)infusion once every 2 weeks, wherein the composition has adose-proportional maximum concentration (C_(max)) and area under curve(AUC) as measurable in the subject's plasma; the AUC_(0-last) of KSPsiRNA is within about 80% to about 125% of (i) 33929.01±18334.75ng*hr/mL or (ii) 28833.57 ng*hr/mL; the C_(max) of KSP siRNA is withinabout 80% to about 125% of (i) 21937.49±7642.00 ng/mL or (ii) 17897.05ng/mL; the AUC_(0-last) of VEGF siRNA is within about 80% to about 125%of (i) 33442.95±18558.98 ng*hr/mL or (ii) 31072.98 ng*hr/mL; and theC_(max) of VEGF siRNA is within about 80% to about 125% of (i)21578.94±6798.07 ng/mL or (ii) 18230.74 ng/mL.
 21. The method of claim19, wherein the subject has cancer.
 22. The method of claim 19, whereinthe subject has advanced cancer with liver involvement.
 23. The methodof claim 19, wherein the subject has endometrial cancer.
 24. The methodof claim 19, wherein the duration of each IV infusion is 15 minutes. 25.The method of claim 19, wherein the duration of each IV infusion 15minutes to 3 hours.
 26. The method of claim 19, wherein the compositionis administered to the subject once every 2 weeks for at least fourweeks.
 27. The method of claim 19, wherein the composition isadministered to the subject once every two weeks for at least 8 weeks.28. The method of claim 19, further comprising administering acorticosteroid to the subject.